I. INTRODUCTION

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The presence of free radicals in biological materials was discovered less than 50 years ago (114). Soon therafter, Denham Harman hypothesized that oxygen radicals may be formed as by-products of enzymic reactions in vivo. In 1956, he described free radicals as a Pandora's box of evils that may account for gross cellular damage, mutagenesis, cancer, and, last but not least, the degenerative process of biological aging (234, 235).

The science of free radicals in living organisms entered a second era after McCord and Fridovich (386) discovered the enzyme superoxide dismutase (SOD) and, finally, convinced most colleagues that free radicals are important in biology. Numerous researchers were now inspired to investigate oxidative damage inflicted by radicals upon DNA, proteins, lipids, and other components of the cell (reviewed in Ref. 49).

A third era began with the first reports describing advantageous biological effects of free radicals. Mittal and Murard (394) provided suggestive evidence that the superoxide anion (O·), through its derivative, the hydroxyl radical, stimulates the activation of guanylate cyclase and formation of the "second messenger" cGMP. Similar effects were reported for the superoxide derivative hydrogen peroxide (615). Ignarro and Kadowitz (272) and Moncada and colleagues (456) discovered independently the role of nitric oxide (NO) as a regulatory molecule in the control of smooth muscle relaxation and in the inhibition of platelet adhesion. Roth and Dröge (472) found that in activated T cells the superoxide anion or low micromolar concentrations of hydrogen peroxide increase the production of the T-cell growth factor interleukin-2, an immunologically important T-cell protein. Keyse and Tyrrell (300) showed that hydrogen peroxide induces the expression of the heme oxygenase (HO-1) gene. Storz and colleagues (551) reported the induction of various genes in bacteria by hydrogen peroxide, and Schreck and Baeuerle (501) reported the activation of the transcription factor nuclear factor B (NF-B) by hydrogen peroxide in mammalian cells.

At the beginning of the 21st century, there is now a large body of evidence showing that living organisms have not only adapted to an unfriendly coexistence with free radicals but have, in fact, developed mechanisms for the advantageous use of free radicals. Important physiological functions that involve free radicals or their derivatives include the following: regulation of vascular tone, sensing of oxygen tension and regulation of functions that are controlled by oxygen concentration, enhancement of signal transduction from various membrane receptors including the antigen receptor of lymphocytes, and oxidative stress responses that ensure the maintenance of redox homeostasis (see Table 1). The field of redox regulation is also receiving growing attention from clinical colleagues in view of the role that oxidative stress has been found to play in numerous disease conditions. These pathological conditions demonstrate the biological relevance of redox regulation. The delicate balance between the advantageous and detrimental effects of free radicals is clearly an important aspect of life. The science of biological "redox regulation" is a rapidly growing field of research that has impact on diverse disciplines including physiology, cell biology, and clinical medicine.

We are now living in a particularly exciting time of redox research where information from different fields and independent approaches is falling into place and beginning to reveal a meaningful picture. This is a good time for a broad overview that summarizes the main principles of redox regulation. In textbook style, this review describes the current knowledge and paradigms but does not discuss future research directions, historical controversies, or experimental models. Moreover, it was not within the scope of this review to deal with all the details. Even the more than 600 references cited here do not cover all relevant publications in the field. For the interested reader, a number of more detailed and specific reviews on this topic are recommended (see Refs. 13, 20, 45, 66, 122, 125, 183, 211, 212, 251, 294, 333, 337, 397, 446, 506, 510, 512, 632, 659).

	II. MAJOR TYPES OF FREE RADICALS AND THEIR DERIVATIVES IN LIVING ORGANISMS

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The superoxide anion is formed by the univalent reduction of triplet-state molecular oxygen (³O₂). This process is mediated by enzymes such as NAD(P)H oxidases and xanthine oxidase or nonenzymically by redox-reactive compounds such as the semi-ubiquinone compound of the mitochondrial electron transport chain (see Fig. 1). SODs convert superoxide enzymically into hydrogen peroxide (130, 187). In biological tissues superoxide can also be converted nonenzymically into the nonradical species hydrogen peroxide and singlet oxygen (¹O₂) (549). In the presence of reduced transition metals (e.g., ferrous or cuprous ions), hydrogen peroxide can be converted into the highly reactive hydroxyl radical (·OH) (101). Alternatively, hydrogen peroxide may be converted into water by the enzymes catalase or glutathione peroxidase (Fig. 1). In the glutathione peroxidase reaction glutathione is oxidized to glutathione disulfide, which can be converted back to glutathione by glutathione reductase in an NADPH-consuming process (Fig. 1).

View larger version (22K): [in this window] [in a new window]   Fig. 1. Pathways of reactive oxygen species (ROS) production and clearance. GSH, glutathione; GSSG, glutathione disulfide.

Because superoxide and NO are readily converted by enzymes or nonenzymic chemical reactions into reactive nonradical species such as singlet oxygen (¹O₂), hydrogen peroxide, or peroxynitrite (ONOO), i.e., species which can in turn give rise to new radicals, the regulatory effects of these nonradical species have also been included in this review. Most of the regulatory effects are indeed not directly mediated by superoxide but rather by its reactive oxygen species (ROS) derivatives. Frequently, different reactive species coexist in the reactive environment and make it difficult to identify unequivocally which agent is responsible for a given biological effect.

The NO radical (NO·) is produced in higher organisms by the oxidation of one of the terminal guanido-nitrogen atoms of L-arginine (437). This process is catalyzed by the enzyme NOS. Depending on the microenvironment, NO can be converted to various other reactive nitrogen species (RNS) such as nitrosonium cation (NO⁺), nitroxyl anion (NO) or peroxynitrite (ONOO) (546). Some of the physiological effects may be mediated through the intermediate formation of S-nitroso-cysteine or S-nitroso-glutathione (207).

The most relevant radicals in biological regulation are superoxide and NO (see Table 1). These radicals are formed by two groups of enzymes, i.e., the NAD(P)H oxidase and NOS isoforms, respectively. Many regulatory effects are mediated by hydrogen peroxide and other ROS that are chemically derived from superoxide.

	III. OXIDATIVE STRESS RESPONSE AS A MODEL OF REDOX SIGNALING

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Free radicals and reactive nonradical species derived from radicals exist in biological cells and tissues at low but measurable concentrations (228, 527). Their concentrations are determined by the balance between their rates of production and their rates of clearance by various antioxidant compounds and enzymes, as illustrated schematically in Figure 2. Halliwell and Gutteridge (228) have defined antioxidants as substances that are able, at relatively low concentrations, to compete with other oxidizable substrates and, thus, to significantly delay or inhibit the oxidation of these substrates. This definition includes the enzymes SOD, glutathione peroxidase (GPx), and catalase, as well as nonenzymic compounds such as -tocopherol (vitamin E), -carotene, ascorbate (vitamin C), and glutathione.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Mechanisms of redox homeostasis. Balance between ROS production and various types of scavengers. The steady-state levels of ROS are determined by the rate of ROS production and their clearance by scavenging mechanisms. Certain antioxidative enzymes including superoxide dismutase (SOD), glutathione peroxidase, catalase, and thioredoxin are potent ROS scavengers but occur in cells only at relatively low concentrations. The same is true for nonenzymic antioxidants. Amino acids and proteins are also ROS scavengers. Amino acids are less effective than the classical antioxidants on a molar basis, but their cumulative intracellular concentration is >0.1 M.

In addition, there are compounds that have a relatively low specific antioxidative activity, i.e., on a molar basis, but, when present at high concentrations, can contribute significantly to the overall ROS scavenging activity. The most prominent examples of such high-level, low-efficiency antioxidants are free amino acids, peptides, and proteins. Practically all amino acids can serve as targets for oxidative attack by ROS, although some amino acids such as tryptophan, tyrosine, histidine, and cysteine are particularly sensitive to ROS (126, 128, 545). Because the cumulative intracellular concentration of free amino acids is on the order of 10¹ M, free amino acids are quantitatively important ROS scavengers (see sect. IIIB5).

Oxygen radicals and other ROS cause modifications of proteins (reviewed in Ref. 220). These oxidative modifications may lead to changes in protein function, chemical fragmentation, or increased susceptibility to proteolytic attack (124, 543, 631). Proteolytic degradation is executed mainly by proteasomes (219). In one of the studies, proteolysis was estimated to increase more than 11-fold after exposure to superoxide or hydrogen peroxide (127). Proteolysis is enhanced by 20-400 µM hydrogen peroxide, whereas millimolar concentrations inhibit proteolysis and may lead to the intracellular accumulation of oxidized proteins (220, 544).

The proteins may differ strongly in their susceptibility to oxidative damage. The redox-sensitive amino acids of bovine serum albumin, for example, were shown to be oxidized about twice as fast as those of glutamine synthase (54), and intact proteins are less sensitive to oxidation than misfolded proteins (161). These findings implicate that 1) phylogenetic evolution has selected for protein structures that are relatively well-protected against oxidation and 2) ROS scavenging activities of intact proteins are weaker than those of misfolded proteins or equivalent concentrations of their constituent amino acids. Protein oxidation and enhanced proteolytic degradation cause, therefore, a net increase in ROS scavenging capacity as schematically illustrated in Figure 2. Preliminary experiments showed that treatment of human skeletal muscle cells with proteasome inhibitors causes a substantial increase in intracellular ROS levels and that this increase is reversed by the addition of free amino acids (R. Breitkreutz and W. Dröge, unpublished observations). More systematic studies are needed to determine the relative contribution of proteins, free amino acids, and classical antioxidant compounds and enzymes to the total ROS scavenging capacity of different cells and tissues.

Living cells and tissues have several mechanisms for reestablishing the original redox state after a temporary exposure to increased ROS or RNS concentrations. The production of NO (NO·), for example, is subject to direct feedback inhibition of NOS by NO (see sect. IIIB1). Elevated ROS concentrations induce in many cells the expression of genes whose products exhibit antioxidative activity (Fig. 2). A major mechanism of redox homeostasis is based on the ROS-mediated induction of redox-sensitive signal cascades that lead to increased expression of antioxidative enzymes or an increase in the cystine transport system, which, in turn, facilitates in certain cell types the increase in intracellular glutathione (see Fig. 2; for details see sect. III, B2-B4). Moreover, because proteins generally provide less ROS scavenging activity than an equivalent amount of the free amino acids contained in them, it is reasonable to assume that oxidative enhancement of proteolysis also contributes, at least to some extent, to the maintenance of redox homeostasis (see Fig. 2 and sect. IIIB5).

Cells or tissues are in a stable state if the rates of ROS production and scavenging capacity are essentially constant and in balance (see Fig. 2 and baseline level in Fig. 3). Redox signaling requires that this balance is disturbed, either by an increase in ROS concentrations or a decrease in the activity of one or more antioxidant systems (Fig. 3). In higher organisms, such an oxidative event may be induced in a regulated fashion by the activation of endogenous RNS- or ROS-generating systems (see sect. IVA). However, similar responses may be induced by oxidative stress conditions generated by environmental factors (see sect. IIIA4). If the initial increase in ROS is relatively small, the antioxidative response may be sufficient to compensate for the increase in ROS and to reset the original balance between ROS production and ROS scavenging capacity. Thus physiological manifestations of redox regulation involve typically a temporary increase and/or a temporary shift of the intracellular thiol/disulfide redox state toward more oxidative conditions, as illustrated in Figure 3. In the long run, these mechanisms tend to maintain a stable state called redox homeostasis.

View larger version (36K): [in this window] [in a new window]   Fig. 3. Regulatory events and their dysregulation depend on the magnitude and duration of the change in ROS or reactive nitrogen species (RNS) concentration. ROS and RNS normally occur in living tissues at relatively low steady-state levels. The regulated increase in superoxide or nitric oxide production leads to a temporary imbalance that forms the basis of redox regulation. The persistent production of abnormally large amounts of ROS or RNS, however, may lead to persistent changes in signal transduction and gene expression, which, in turn, may give rise to pathological conditions.

Under certain conditions, however, ROS production is increased more strongly and persistently, and the antioxidative response may not be sufficient to reset the system to the original level of redox homeostasis. In such cases, the system may still reach an equilibrium according to the model in Figure 2, but the resulting quasi-stable state may now be associated with higher ROS concentrations and different levels of free amino acids and/or different patterns of gene expression due to redox-sensitive signaling pathways. Indications for such a shift to more oxidative conditions have been seen in the process of aging (see sect. VIC), implying that a pro-oxidative shift may not always be associated with an overtly pathological condition.

Pathological conditions (see sect. VI, H-P) may develop in more extreme cases of persistently high ROS levels (see Fig. 3). Again, these conditions do not necessarily involve a loss of homeostasis but rather a chronic shift in the level of homeostasis. Accordingly, pathological symptoms may result from both the damaging effects of ROS and from ROS-mediated changes in gene expression.

In several cases (see Table 2), changes in the intracellular thiol/disulfide redox state have been shown to trigger the same redox-responsive signaling proteins and pathways as those triggered by hydrogen peroxide (28, 192, 248, 323). Bacterial OxyR (see sect. IIIB2) is a model case of a redox-sensitive signaling protein that may be activated either directly by hydrogen peroxide or, alternatively, by changes in the intracellular glutathione redox state (28). Conversely, protein tyrosine phosphatases (see sect. VB) are inactivated either by ROS or by pro-oxidative changes in the intracellular thiol/disulfide redox state. Accordingly, this review also addresses the role of the thiol/disulfide redox state in the control of cell function.

Certain alkylating agents play an important role as environmental carcinogens by exerting effects on redox-sensitive signaling pathways similar to those induced by ROS (621). There is a strong possibility that the resulting dysregulation of signaling cascades may contribute to the process of carcinogenesis (see sect. VII).

Certain heavy metal compounds are important environmental allergens. A point in case is the sulfhydryl-reactive compound mercury dichloride (HgCl₂). Treatment of murine lymphocytes with HgCl₂ was found to induce strong aggregation and activation of the protein tyrosine kinase p56^lck. These findings suggest that the oxidation of redox-reactive sulfhydryl groups of certain signaling proteins leads to a dysregulation of lymphocytes and may contribute thereby to the development of allergies (411).

These examples illustrate that the mechanisms of redox regulation may be targets for hazardous environmental agents. However, a more detailed discussion of this point would exceed the scope of this review.

Many of the enzymes that utilize a heme prosthetic group in catalysis are inactivated by NO. This applies to the heme-containing cytochrome P-450-related enzyme NOS, leading to feedback inhibition of NO production by NO (6, 79, 215). The inhibition of NOS was shown to involve the formation of a ferrous-nitrosyl complex (6).

The protective responses of bacteria against ROS are among the best investigated examples of redox-regulated gene expression (reviewed in Ref. 658). Prokaryotes have several different signaling pathways for responding to ROS or to alterations in the intracellular redox state (28, 45, 176, 551, 634, 657). Studies in Escherichia coli revealed that low levels of ROS activate the expression of several gene products involved in the antioxidant defense including Mn-SOD (238), catalase (649), hydroperoxidase I (katG), an alkylhydoperoxide reductase (ahpCF), glutathione reductase (gorA), glutaredoxin 1 (grxA), and a regulatory RNA (oxyS).

At least nine proteins that are synthesized in Salmonella typhimurium and E. coli after exposure to hydrogen peroxide are under the control of the oxyR locus (45, 108, 138). The OxyR protein controls protective responses against normally lethal doses of hydrogen peroxide or against killing by heat. In addition, it negatively autoregulates its own expression (109, 489). Hydrogen peroxide does not stimulate the synthesis of OxyR but converts the reduced form of OxyR into its oxidized and regulatory competent form (Fig. 4) (45, 551, 657). Exposure of the OxyR protein to hydrogen peroxide results initially in the conversion of Cys-199 to a sulfenic acid derivative which subsequently forms an intramolecular disulfide bond with Cys-208, as illustrated schematically in Figure 4 (657). Because the redox potential of OxyR is 185 mV, and the thiol-disulfide redox state of the normal bacterial cytoplasm is typically 260 to 280 mV, the OxyR is normally in the reduced form and exemplifies proteins that can be activated either through direct oxidation by hydrogen peroxide or by an oxidative shift in the thiol/disulfide redox status, as illustrated in Figure 4 (28). Both oxidized and reduced OxyR are able to bind to the oxyR-oxyS promoter region but exhibit different binding characteristics (578). The formation of disulfide bonds can be reversed by glutaredoxin 1 and by thioredoxin (Trx) (657). Because OxyR controls the induction of glutathione reductase and glutaredoxin 1, the OxyR response is part of an autoregulatory circuit.

View larger version (17K): [in this window] [in a new window]   Fig. 4. Schematic model of OxyR activation. The regulatory protein OxyR is activated by the formation of disulfide bridges. This process is mediated either by hydrogen peroxide (H2O2) or by oxidative changes in the intracellular thiol/disulfide redox state.

Protective responses against superoxide are controlled by the sox locus (255, 581), which regulates the induction of ~10 proteins, including Mn-SOD, NADPH:ferredoxin oxidoreductase, and glucose-6-phosphate dehydrogenase (reviewed in Ref. 45). One of the gene products from the sox locus, the SoxR protein, exists in solution as a homodimer containing two stable (2Fe-2S) centers that are anchored to four cysteine residues near the COOH terminals (67, 255, 634). Under normal physiological conditions, these iron-sulfur centers are in the reduced state. They are readily oxidized, however, under oxidative stress (149). The oxidative process is reversible if the oxidative stress conditions are removed. Only the oxidized form of SoxR stimulates transcription of soxS (67, 149, 195). Both the oxidized and the reduced forms of SoxR can bind to DNA but interact differently with RNA polymerase.

The redox-sensitive proteins OxyR and SoxR have been described here as two particularly prominent and well-investigated cases of bacterial redox regulation. Additional examples of redox-responsive regulatory mechanisms in prokaryotic cells have been reviewed by Bauer et al. (45).

Treatment of S. cerevisiae with hydrogen peroxide activates the yAP-1 transcription factor that binds specifically to the AP-1 site of the eukaryotic AP-1 family of transcription factors (323). Similar activation can be induced by diamide or diethyl maleate, i.e., two thiol oxidants that modulate the intracellular reduced glutathione (GSH) state. The yAP-1 transcription factor is involved in protective responses against oxidative stress, inducing Trx production from the TRX2 gene (323). Overexpression of yAP-1 on a multicopy plasmid was also found to increase the expression of SOD, glutathione reductase, and glucose-6-phosphate dehydrogenase, whereas yAP-1 mutant strains showed greatly decreased levels of these enzymes (498). Oxidative activation of yAP-1 operates at the posttranslational level and involves the translocalization of yAP-1 from the cytoplasm to the nucleus. This translocation is controlled by a cysteine-rich domain at the COOH terminus. Three conserved cysteine residues in this region are believed to be important in sensing the redox state (324). The transcription factor SKN7 cooperates with yAP-1 and is, therefore, also critically involved in oxidative stress response (399).

The response of S. cerevisiae to hydrogen peroxide (0.4 mM for 15 min) was studied by two-dimensional gel electrophoresis of total cell proteins, revealing an increase in the expression of 115 proteins and a decrease in the expression of another 52 proteins (200). The induced proteins include Mn-SOD, Cu/Zn-SOD, glutathione reductase, catalase, Trx reductase, cytochrome-c peroxidase, several proteasome subunits, and heat shock proteins. In addition, carbohydrate metabolism is rapidly redirected to the regeneration of NADPH at the expense of glycolysis.

The redox-responsive signal cascades in mammalian cells show certain similarities to the oxidative stress response of S. cerevisiae. While activated macrophages and neutrophils in inflamed tissue of higher organisms generate massive amounts of ROS to kill environmental pathogens (see sect. IVA2), other host cells must be protected against this oxidative burst. Lymphocytes recruited into the inflammatory environment initiate antigen-specific immunological effector mechanisms and can function only because they are able to activate powerful protective mechanisms against the oxidative stress. Exposure to ROS or changes in the intracellular thiol redox state modulate various signal transduction cascades and increase the activities of several transcription factors (reviewed in Refs. 12, 13, 183, 337, 397, 446, 506, 512).

The oxidoreductase Trx is one of the proteins that is inducibly expressed in lymphocytes and other cells by hydrogen peroxide, ultraviolet (UV) irradiation, and other conditions of oxidative stress (373, 480, 565). Together with the glutathione system, Trx plays a key role in the maintenance of a reducing intracellular redox state in higher organisms. The 5'-upstream sequence of the human Trx gene contains putative binding sites for the redox-responsive transcription factors AP-1 and NF-B (407, 408).

Exposure of macrophages to low levels of ROS or other inducers of oxidative stress induces the expression of peroxyredoxin I (i.e., a Trx peroxidase), heme oxygenase-1 (HO-1), and the cystine transporter x (278, 486). Because the plasma concentration of reduced cysteine is relatively low, the cystine transporter plays a limiting role in the cellular supply of cyst(e)ine and in the biosynthesis of glutathione in macrophages and lymphocytes (reviewed in Ref. 160). ROS and various other oxidants were also found to induce MnSOD mRNA levels to a moderate extent in several cell types (522).

The redox control of the heme oxygenase-1 (HO-1) gene is one of the best studied models of redox regulation in mammalian cells. HO-1 induction in skin fibroblasts may serve as an inducible defense pathway to remove heme liberated by oxidants. The HO-1 protein and mRNA are strongly induced by ROS, physiological doses of UVA irradiation, and various other inducers of oxidative stress, including NO (237, 300, 585, 586). The UVA response is synergistically enhanced by depletion of intracellular glutathione. The sustained induction of HO-1 mRNA and its inducibility in many tissues and various mammalian species has rendered HO-1 mRNA a useful marker for cellular oxidative stress at the mRNA level. In murine macrophages, HO-1 expression is induced by hydrogen peroxide via AP-1 (16, 91, 92). Activation of ERK and p38 MAPK was implicated in HO-1 expression in chicken hepatoma cells (167).

	IV. NITRIC OXIDE AND REACTIVE OXYGEN SPECIES AS REGULATORY MEDIATORS OF PHYSIOLOGICAL RESPONSES

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The following sections describe cases of redox regulation that serve physiological functions other than protection against oxidative stress and redox homeostasis. These cases typically involve the regulated production of NO or ROS and the resulting effects on defined signaling cascades.

A.  Regulated Production of Free Radicals in Higher Organisms

1.  Regulated production of NO

The enzyme NOS exists in three isoforms, i.e., neuronal NOS (nNOS; type I) (69), inducible NOS (iNOS; type II) (639), and endothelial NOS (eNOS; type III) (330). Many tissues express one or more of these isoforms. The isoforms nNOS and eNOS are constitutively expressed, but their activity is regulated by the intracellular calcium concentration. The isoform iNOS is inducibly expressed in macrophages after stimulation by cytokines, lipopolysaccharides, and other immunologically relevant agents (reviewed in Ref. 59). Expression of iNOS is regulated at the transcriptional and posttranscriptional level by signaling pathways that involve agents such as the redox-responsive transcription factor NF-B or mitogen-activated protein kinases (MAPKs) (366). The rate of NO synthesis is also determined to some extent by the availability of the substrate L-arginine and by the cofactor tetrahydrobiopterin (BH₄).

Activated macrophages and neutrophils can produce large amounts of superoxide and its derivatives via the phagocytic isoform of NADPH oxidase. This enzyme is a heme-containing protein complex illustrated schematically in Figure 5. In an inflammatory environment hydrogen peroxide is produced by activated macrophages at an estimated rate of 2-6 × 10¹⁴ mol·h¹·cell¹ and may reach a concentration of 10-100 µM in the vicinity of these cells (299, 335, 412). The massive production of antimicrobial and tumoricidal ROS in an inflammatory environment is called the "oxidative burst" and plays an important role as a first line of defense against environmental pathogens. The physiological relevance of NADPH oxidase as a defense agent is suggested by the observation that mice lacking the NADPH oxidase components gp91^phox or p47 exhibit reduced resistance to infection (132, 148, 169, 400, 448, 466, 521). The combined activities of NADPH oxidase and myeloperoxidase in phagocytes leads, in addition, to the production of hypochlorous acid (HClO), one of the strongest physiological oxidants and a powerful antimicrobial agent (229, 500). Stimulated neutrophils and macrophages generate also singlet oxygen by reactions that involve either myeloperoxidase or NADPH oxidase (549). Importantly, however, physiologically relevant ROS concentrations can also modulate redox-sensitive signal cascades and enhance immunological functions of lymphocytes (see sect. IVF).

View larger version (22K): [in this window] [in a new window]   Fig. 5. Structure of neutrophil NAD(P)H oxidase. The enzyme consists of the membrane-bound cytochrome b558 complex comprising gp91phox and p22phox, the cytosolic proteins p47 and p67, and a low-molecular-weight G protein of the rac family.

Phagocytic NADPH oxidase becomes activated upon translocation of cytosolic p47, p67, and a G protein of the rac family to the membrane-bound cytochrome b₅₅₈ complex that contains gp91^phox and p22 (Fig. 5). The catalytic moiety gp91^phox is a plasma membrane-associated complex protein containing a flavin-adenine dinucleotide component and two hemes (for details see Refs. 211, 333). The activation of phagocytic NADPH oxidase can be induced by microbial products such as bacterial lipopolysaccharide, by lipoproteins, or by cytokines such as interferon-, interleukin-1, or interleukin-8 (60). The activation of NADPH oxidase is mainly controlled by the rac isoform rac2 in neutrophils and rac1 in macrophages and monocytes (567, 346).

The production of ROS by nonphagocytic NAD(P)H oxidase isoforms plays a role in the regulation of intracellular signaling cascades in various types of nonphagocytic cells including fibroblasts, endothelial cells, vascular smooth muscle cells, cardiac myocytes, and thyroid tissue (36, 163, 210, 211, 288-290, 354, 387, 553, 557, 559, 571, 572, 660). In most of these cases, rac1 is involved in the induction of NAD(P)H oxidase activity (289, 290, 660). Muscle cells and fibroblasts account for most of the superoxide produced in the normal vessel wall.

The NAD(P)H oxidase isoforms of the cardiovascular system are membrane-associated enzymes that appear to utilize both NADH and NADPH (211). The rate of superoxide production in nonphagocytic cells is only about one-third of that of neutrophils. Vascular smooth muscle cells, in contrast to neutrophils, endothelial cells, or fibroblasts, generate superoxide and hydrogen peroxide mainly intracellularly.

The cardiovascular NAD(P)H oxidase isoforms are induced by hormones, hemodynamic forces, or by local metabolic changes (211). Angiotensin II increases NAD(P)H-driven superoxide production in cultured vascular smooth muscle cells and fibroblasts. Thrombin, platelet-derived growth factor (PDGF), and tumor necrosis factor- (TNF-) stimulate NAD(P)H oxidase-dependent superoxide production in vascular smooth muscle cells. Interleukin-1, TNF-, and platelet-activating factor increase NAD(P)H-dependent superoxide production in fibroblasts. Mechanical forces stimulate NAD(P)H oxidase activity in endothelial cells. Reoxygenation stimulates NAD(P)H oxidase activity in cardiac myocytes.

Whereas the gp91^phox of phagocytic cells (see Fig. 5) has also been found in endothelial cells, other nonphagocytic cells appear to utilize structural homologs of gp91^phox. Several novel homologs of gp91^phox have recently been identified and are the subject of intense research. The gp91^phox homolog p138^tox is (the catalytic unit of) the NADPH oxidase that supports thyroid hormone biosynthesis (163). An NAD(P)H oxidase with low affinity for oxygen and high affinity for cyanide is believed to act as one of the sensors for oxygen tension in the carotid body; these sensors control the rate of ventilation (8). The function of oxygen sensing is apparently shared by several proteins, including a nonmitochondrial cytochrome b₅₅₈, a mitochondrial protein, and possibly a third heme protein (329, 659). A similar group of proteins was suggested to be involved as oxygen sensors in the regulation of erythropoietin production in human hepatoma cells (659). A microsomal NADH oxidase was implicated as an oxygen sensor in bovine pulmonary and coronary arteries, where changes in oxygen tension regulate vascular relaxation through changes in superoxide production and cGMP formation (632).

There is a strong possibility that rac-like proteins also occur in plants (5, 624), where they may be involved in the induction of NAD(P)H oxidase-like enzymes (569). The oxidative burst in plants is an effective bactericidal mechanism.

The enzyme 5-lipoxygenase (5-LO) has been identified as an inducible source of ROS production in lymphocytes (60, 359), but the evidence for its physiological role in redox signaling is still scarce. Lipoxygenases are non-heme-containing dioxygenases that oxidize polyunsaturated fatty acids at specific carbon sites to give hydroperoxy fatty acid derivatives with conjugated double bonds. The numbers in specific enzyme names such as 5-LO, 12-LO, or 15-LO refer to the arachidonic acid site that is predominantly oxidized (644). 5-LO is best known for its role in the biosynthesis of the leukotrienes A₄, B₄, C₄, D₄, and E₄. The oxidized metabolites generated by 5-LO were found to change the intracellular redox balance and to induce signal transduction pathways and gene expression. 5-LO was shown to be involved in the production of hydrogen peroxide by T lymphocytes after ligation of the CD28 costimulatory receptor (359) and in response to interleukin-1 (60). Hydrogen peroxide production in response to CD28 stimulation was found to be decreased by specific inhibitors of 5-LO or lipid peroxidation but not by inhibitors of NADPH oxidase or cyclooxygenase. The physiological role of 5-LO in CD28-mediated signal transduction remains to be confirmed by more detailed studies.

Cyclooxygenase-1 has been implicated in ROS production in cells stimulated with TNF-, interleukin-1, bacterial lipopolysaccharide, or the tumor promoter 4-O-tetradecanoylphorbol-13-acetate (TPA) (178, 406). A role for cyclooxygenase in the inflammatory response of 5-LO-defective mice was also suggested (206); however, the evidence for the participation of cyclooxygenase in redox signaling is still scarce.

Guanylate cyclase belongs to the family of heterodimeric heme proteins and catalyzes the formation of cGMP, which is utilized as an intracellular amplifier and second messenger in a large range of physiological responses (197). NO binds to the heme moiety of guanylate cyclase, disrupting the planar form of the heme iron. The resulting conformational change activates the enzyme (273). Its product cGMP modulates the function of protein kinases, phosphodiesterases, ion channels, and other physiologically important targets (497). The most important examples include the regulation of smooth muscle tone (272) and the inhibition of platelet adhesion (456). Vascular smooth muscle relaxation is mediated by a cGMP-dependent protein kinase that phosphorylates and activates a calcium-sensitive potassium channel (27). Other examples have been reviewed by Deora and Lander (140).

Superoxide and hydrogen peroxide may also play a role in the activation of guanylate cyclase (82, 394, 615). Since micromolar concentrations of hydrogen peroxide stimulate NOS (190), there is a strong possibility that the activation of guanylate cyclase by superoxide and hydrogen peroxide may be mediated, at least in part, indirectly via NOS. Other studies suggest that compound I, a form of catalase, may play a critical role in the activation of guanylate cyclase by hydrogen peroxide (82). After interacting with hydrogen peroxide, catalase is converted into an oxidized heme intermediate called compound I, which is normally converted back into its reduced form by a second molecule of hydrogen peroxide.

In iron-sulfur proteins, iron is bound simultaneously to inorganic sulfide groups and cysteine thiolate groups of the proteins. Such proteins are sensitive to both ROS and RNS. Oxidation usually results in dissolution of the iron-sulfur cluster and loss of function (85, 97, 250).

A special case is mammalian (4Fe-4S) aconitase, which is involved in the citric acid cycle. RNS inhibit the aconitase activity by disrupting the Fe-S clusters but expose simultaneously an RNA-binding site with specificity for the iron-response elements of the transferrin receptor and ferritin mRNAs. In this form the protein is called iron-regulatory protein-1 and is involved in iron homeostasis. In this case, the RNS-induced loss of one enzyme function is associated with the gain of or switch to another function for the same protein.

In human peripheral blood mononuclear cells and endothelial cells, NO was found to activate all three MAPK pathways (89, 336, 339, 445). The effect has been attributed to the NO-mediated stimulation of a membrane-associated protein tyrosine phosphatase activity which may lead to the dephosphorylation and activation of the Src family protein tyrosine kinase p56^lck (339). Another Src family protein kinase, p60^c-src, was also found to be activated by NO in fibroblasts or immunoprecipitates (15). The activation was associated with autophosphorylation at Tyr-416 and S-S bond-mediated aggregation of the kinase molecules. NO may also activate Ras by S-nitrosylation of cysteine-118 (335-338).

Oxygen homeostasis is maintained in higher organisms by a tight regulation of the red blood cell mass and respiratory ventilation. Carotid bodies are sensory organs that detect changes in arterial blood oxygen. They are composed of glomus type I chemoreceptor cells that release neurotransmitters in response to hypoxia. This process changes the level of electrical activity in the efferent fibers of the carotid sinus nerve, thus relaying the sensory information to the brain stem neurons that regulate breathing. A growing body of evidence indicates that changes in oxygen concentration are sensed independently by several different ROS-producing proteins including a b-type cytochrome with properties similar to those of the cytochrome b₅₅₈ in the NADPH oxidase complex in neutrophils (reviewed in Ref. 7). Other studies suggest that changes in the rate of mitochondrial ROS production may play a major role in oxygen sensing by the carotid body (80, 449). Furthermore, it was found that the -subunit of the potassium channel resembles the structure of NADPH-oxidoreductase (221). It is widely accepted that transduction of the sinus nerve signal involves changes in the K⁺-channel conductivity of type I cells in response to changes in oxygen tension (356). Exactly how changes in ROS production are translated into changes in K⁺ channel activity remains to be established. Acker and Xue (9) implicated ROS-mediated changes in the glutathione redox state in the control of K⁺ efflux and the corresponding Ca²⁺ influx.

The red blood cell mass is regulated by the hormone erythropoietin, which is mainly produced by kidney and liver cells, following stimulation by hypoxia. The oxygen-sensing mechanisms are still unclear and the subject of controversy. It is clear, however, that changes in oxygen tension are sensed by changes in ROS production (174, 264, 291, 413). Expression of erythropoietin protein or mRNA in hepatoma cell lines or perfused kidneys was found to be strongly repressed by hydrogen peroxide. Treatment of normoxic cells with exogenous catalase stimulated erythropoietin production (94). A cytochrome b-like NAD(P)H oxidase is believed to play a role as a major oxygen sensor and ROS producer. However, the role of a mitochondrial mechanism of ROS production has also been reported (7, 103, 173, 659).

The erythropoietin gene is controlled by the transcription factor hypoxia-inducible factor 1 (HIF-1) (608) (as illustrated schematically in Fig. 6). HIF-1 is a heterodimeric protein composed of the subunits HIF-1 and HIF-1 (Fig. 6). The HIF-1 and HIF-1 genes are constitutively expressed, and changes in oxygen tension fail to affect the concentration of the HIF-1 subunit. In contrast, under normoxic conditions, HIF-1 is rapidly degraded by proteasomes in an ROS-dependent manner (265). Hypoxia decreases the ROS-mediated degradation of HIF-1 and enhances thereby the formation of the heterodimeric complex (Fig. 6) (510, 659).

View larger version (28K): [in this window] [in a new window]   Fig. 6. Regulation of the transcription factor hypoxia-inducible factor 1 (HIF-1). HIF-1 is a heterodimeric protein composed of the subunits HIF-1 and HIF-1, the genes of which are both constitutively expressed. Changes in oxygen tension fail to affect the concentration of HIF-1, whereas HIF-1 is rapidly degraded under normoxic conditions by proteasomes in a ROS-dependent fashion. In these cells, ROS production is tightly linked to oxygen concentrations and, therefore, serves as a sensor for oxygen tension. The figure illustrates ROS production by a membrane-bound NAD(P)H oxidase.

HIF-1 and HIF-1 mRNAs are expressed in most if not all human and rodent tissues (510). The number of target genes activated by HIF-1 continues to increase, and targets include genes whose protein products are involved in angiogenesis, energy metabolism, erythropoiesis, cell proliferation and viability, vascular remodeling, and vasomotor responses (reviewed in Ref. 510).

The same transcription factor is now known to control the production of a variety of hypoxia-regulated hormones and proteins including the vascular endothelial growth factor (VEGF) that stimulates the formation of new blood vessels (80) and the tyrosine hydroxylase (TH) that facilitates the control of ventilation by the carotid body (reviewed in Refs. 510, 659).

Controlled changes in the adhesive properties of cells and tissues play an important role in many biological processes. Adhesion of leukocytes to endothelial cells in postcapillary venules, for example, is an early step in chronic inflammation and depends on the expression of cell-surface receptors known as cell adhesion molecules (17). Cell adhesion molecules are also implicated in embryogenesis, cell growth, differentiation, and wound repair (186). The expression of cell adhesion molecules is stimulated by bacterial lipopolysaccharides and by various cytokines such as TNF, interleukin-1, and interleukin-1 (17).

The adherence of leukocytes to endothelial cells is also induced by ROS (478, 509). This effect is abolished by catalase but not by superoxide dismutase, suggesting that hydrogen peroxide and not superoxide is the effective agent (509). Moreover, the oxidant-induced adherence of neutrophils is inhibited by hydroxyl radical scavengers or iron chelators, suggesting that the induction of adherence may be mediated by hydroxyl radicals generated from hydrogen peroxide within the cell.

Adhesion of neutrophils to endothelial cells involves the intercellular adhesion molecule-1 (ICAM-1), CD11b/CD18, and L-selectin (509). In addition, ROS treatment of endothelial cells induces the phosphorylation of the focal adhesion kinase pp125^FAK, a cytosolic tyrosine kinase that has been implicated in the oxidant-mediated adhesion process (477, 488).

Lymphocytes are the carriers of immunological specificity and, therefore, play an important role in the defense against environmental pathogens. A sophisticated combination of regulatory mechanisms ensures that even minute amounts of pathogen activate highly aggressive responses without causing major damage to host tissue. The immune response typically involves the lymphocyte receptor for antigen, receptors for costimulatory signals, and various types of cytokine (507, 516). The response is also subject to regulation by redox processes. The functional activation of T lymphocytes is strongly enhanced by ROS and/or by a shift in the intracellular glutathione redox state (248). Superoxide and/or physiologically relevant concentrations of hydrogen peroxide were shown to augment the production of interleukin-2 by antigenically or mitogenically stimulated T cells in various experimental systems (358, 472). Low micromolar concentrations of hydrogen peroxide were also shown to induce the expression of the interleukin-2 receptor in a mouse T-cell lymphoma line (358).

T-cell functions such as interleukin-2 production can be readily induced in vitro without the addition of ROS simply by exposure of T cells to relatively high concentrations of antigen or antibodies that bind to the T-cell receptor and the costimulatory receptor CD28 (352). In T cells, strong activation of the costimulatory receptor CD28 causes a significant decrease in intracellular glutathione levels and the endogenous production of hydrogen peroxide (359). An intact organism, however, is typically infected by relatively small concentrations of pathogens, and ligand concentrations for the T-cell receptor and the costimulatory CD28 receptor are expected to be suboptimal, at least in the initial phases of the infection.

Exposure of T lymphocytes to physiologically relevant concentrations of environmental ROS or other inducers of moderate oxidative stress does not bypass the requirement for signaling cascades initiated by specific cell membrane receptors, but such exposure can amplify signaling cascades after relatively weak receptor stimulation (248). Signaling cascades from the various cell membrane receptors are differentially regulated by ROS. Transcription from the interleukin-2 promoter is strongly enhanced in Jurkat T cells by exposure to 50 µM hydrogen peroxide in combination with but not without anti-CD28 ligands, indicating that the redox effect can enhance the stimulatory signal from the antigen receptor but cannot replace the signal from the CD28 costimulatory receptor. Thus hydrogen peroxide from the inflammatory environment thus appears to decrease the triggering thresholds of the antigen receptor-dependent signal cascades as illustrated schematically in Figure 7. It is often critical for the survival of the infected host that a specific immune response be induced before optimal antigen doses have accumulated.

View larger version (40K): [in this window] [in a new window]   Fig. 7. Functions of ROS in the immunological response against environmental pathogens. The massive production of ROS (oxidative burst) by activated macrophages in the inflammatory environment provides a first line of defense against environmental pathogens. A certain fraction of pathogens, however, may escape this rapid but moderately effective manifestation of "innate immunity" and may generate within a few days a large progeny of pathogens. Antigenic peptides generated within the activated macrophages by the breakdown of pathogens are presented by major histocompatibility complex (MHC) determinants to the antigen receptors (AR) of T lymphocytes. This interaction triggers the proliferation and differentiation of the T cells and leads within a few days to a large progeny of immunological effector cells. The effector cells provide a highly effective and antigen-specific immunological defense. ROS that are concomitantly produced by the activated macrophages in the inflammatory environment enhance the AR-mediated signal cascades and decrease thereby the activation threshold of the T cells. Without this effect, the T lymphocytes would require relatively large concentrations of antigenic peptides and would lose valuable time in their "race" with the proliferating pathogens. In this situation time may be a matter of life or death for the organism.

The importance of the oxidative microenvironment in the activation of immune responses by small concentrations of antigen in vivo is exemplified by studies of the generation of cytotoxic T lymphocytes in mice after in vivo immunization with small numbers of syngeneic tumor cells or with cells expressing foreign minor histocompatibility antigens (474). The simultaneous injection of glutathione was found to enhance immunization with relatively large numbers of stimulator cells but suppress in vivo immunization by small numbers of stimulator cells (i.e., small amounts of antigen). The glutathione-dependent enhancement of the response to high doses of antigen is in line with the requirement for reducing conditions in the execution phase of the signaling cascade and for the DNA synthesis (see sect. VK) and suggests that this requirement may become a limiting factor under conditions of high antigen concentrations.

Last but not least, there is evidence that the intracellular redox state also modulates the immunological functions of macrophages. Hamuro et al. (230) reported that macrophages vary strongly in their release of prostaglandins, interleukin-6, and interleukin-12, depending on the intracellular content of glutathione. The balance between "reductive" and "oxidative" macrophages regulates thereby the ratio of helper T cells of type 1 versus type 2 (TH1/TH2).

G.  Role of ROS in Programmed Cell Death

1.  Induction and execution of apoptosis

Apoptosis is a special form of programmed cell death that plays an indispensable role in the development and homeostasis of multicellular organisms (636). An increase in cellular ROS production is often observed in apoptotic processes triggered by various stimuli including APO-1/Fas/CD95 ligands (41, 171, 257, 287, 317, 588, 623, 655). However, some authors found that triggering of the APO-1/Fas/CD95 receptor does not induce ROS production (268), and some authors observed membrane changes typical of apoptosis in the absence of ROS (96, 281), indicating that pro-oxidative conditions are not a general prerequisite for apoptotic cell death. Nevertheless, high ROS concentrations induce apoptotic cell death in various cell types (reviewed in Refs. 162, 535), suggesting that ROS contribute to cell death whenever they are generated in the context of the apoptotic process. Exposure of T lymphocytes to relatively moderate concentrations of hydrogen peroxide was found to induce a CD95-independent apoptotic process that requires mitochondrial ROS production and the activation of NF-B (162). This underscores the need to transfer lymphocytes to more reducing conditions for the development of immunological effector functions (see sect. VK).

NO-dependent apoptosis has been observed in several experimental models and certain clinical pathologies (10, 18, 76, 132, 575). Induction of apoptosis by NO is associated with a decrease in the concentration of cardiolipin, decreased activity of the mitochondrial electron transport chain, and release of mitochondrial cytochrome c into the cytosol (589, 592). However, some cell types, such as endothelial cells from the microvasculature, are extremely resistant to the induction of apoptosis by NO (357), and low concentrations of NO provide protection from apoptotic cell death in various cell types by inhibiting certain caspases (113, 304, 349, 479). High intracellular glutathione levels are associated with increased resistance to NO-mediated apoptosis (589).

TNF- induces cell death in many types of tumor cells and has been used in model systems for studies of the molecular mechanisms of cell death. In transformed cell lines TNF- induces endogenous ROS production by mitochondria (423, 504). Whether and how these ROS contribute to the induction of cell death depends on the signaling and execution pathways that are activated (145). In leukocytes and fibroblasts, TNF- induces the release of superoxide by the activation of membrane-bound NADPH oxidases. This process induces proliferation or cell death depending on the condition of the ROS-producing cell (249, 310, 387, 504, 515).

The attempted infection of plants by an avirulent pathogen was found to induce a protective "hypersensitive response" (HR) that involves the generation of superoxide and hydrogen peroxide (331). These ROS induce a number of defense genes or drive cells into apoptosis (279, 331, 347). A study of the infection of Arabidopsis leaves by avirulent Pseudomonas syringae revealed that the local primary oxidative burst leads to subsequent secondary microbursts in distant leaves and contributes thereby to the development of a state of systemic acquired resistance (SAR) (21). Activation of SAR is associated with the expression of several gene families, i.e., the pathogenesis-related (PR) genes (587). Salicylic acid acts as a signaling molecule in the system (617). The receptor for salicylic acid has catalase activity (105). Binding of salicylic acid causes the downregulation of catalase activity and leads thereby to a significant increase in the intracellular hydrogen peroxide concentration at sites distant to the original insult (105). The distant accumulation of (secondary) low levels of hydrogen peroxide induces the expression of various defense genes and gene products including chitinase and peroxidase (633). Two stress MAPKs of Arabidopsis, i.e., AtMPK3 and AtMPK6, are activated by hydrogen peroxide through the MAPK kinase ANP1 (318).

A particularly active field of research deals with the role of ROS in the regulation of cardiovascular functions. Practically all cell types in the vascular wall produce ROS. Upon stimulation by growth factors or cytokines, vascular NAD(P)H oxidases produce superoxide and other ROS that activate multiple intracellular signaling pathways. Thus ROS play a decisive role in the normal functioning of cardiac and vascular cells (211, 559).

The regulation of vascular tone by cGMP is a special case. The enzyme guanylate cyclase was found to be activated by both NO and hydrogen peroxide (272, 394, 615, 632). This effect of hydrogen peroxide is believed to play a role in the oxygen-dependent regulation of cGMP levels and vascular contraction.

Other responses to changes in oxygen tension include the control of ventilation by the carotid body and the regulated production of certain hormones such as erythropoietin, VEGF, and insulin-like growth factor II (IGF-II). All of these hormones are under transcriptional control of the transcription factor HIF-1 (510, 659).

One of the well-studied cell types with respect to redox-responsive signaling cascades is the lymphoid cell. The redox sensitivity of antigen-induced signaling cascades strongly suggests that massive ROS production by phagocytic cells in the inflammatory environment (i.e., the oxidative burst) serves not only as a first line of defense against environmental pathogens but also enhances the response of lymphocytes to (small amounts of) antigen.

	V. REDOX-SENSITIVE TARGETS IN SIGNALING CASCADES

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There are various examples of growth factors, cytokines, or other ligands that trigger ROS production in nonphagocytic cells through their corresponding membrane receptors (see sect. IVA3). Such ROS production can mediate a positive feedback effect on signal transduction from these receptors since intracellular signaling is often enhanced by ROS or by a pro-oxidative shift of the intracellular thiol/disulfide redox state, as illustrated in Figure 8 (36, 39, 50, 115, 301, 354, 355, 525, 554, 557, 558, 571, 606, 635). The molecular details of the oxidative enhancement are not entirely clear. However, work on different receptors and signaling pathways revealed certain consistent patterns that have important physiological implications.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Role of ROS in epidermal growth factor (EGF) receptor-mediated signaling. The interaction of the EGF receptor or other related membrane receptors with corresponding ligands leads to activation of NAD(P)H oxidase and the production of superoxide and hydrogen peroxide. Hydrogen peroxide, in turn, facilitates the autophosphorylation of the membrane receptor and the induction of the signal cascade. The activation of NAD(P)H oxidase by other membrane receptors such as the angiotensin II receptor can provide a cooperative effect that contributes to the autophosphorylation of the EGF receptor and to the activation of the EGF-dependent signaling cascade.

For example, the role of ROS has been demonstrated for nerve growth factor (NGF) signaling in neuronal cells (558), for epidermal growth factor (EGF) signaling in human epidermoid carcinoma cells (36), and for PDGF (37, 246). Stimulation by any of these growth factors results in a transient increase in intracellular ROS through the signaling protein Rac1. Elimination of hydrogen peroxide by catalase was shown to inhibit EGF- and NGF-induced tyrosine phosphorylation of various cellular proteins, including phosphorylation of the growth factor receptor itself (see Fig. 8). In the case of the EGF receptor, induction of ROS production was reported to require the kinase activit