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Neuroscientist. 11:583–594. van der Walt JM, Nicodemus KK, Martin ER et al. 2003. Mitochondrial polymorphisms signi cantly reduce the risk of Parkinson’s disease. Am J Hum Genet. 72:804–811. 473 Chapter 19 REDOX SIGNALING AND VASCULAR FUNCTION J. Will Langston, Magdalena L. Circu, and Tak Yee Aw ABSTRACT Over the last two decades, redox signaling has emerged as an important regulator of cell function, and it is now well appreciated that reactive oxygen and nitrogen species act as second messengers that modulate vascular activity via direct interactions with specifi c enzymes, proteins, and transcription factors to regulate cell signaling and/or gene expression. The growing interest in the role of redox signaling in the vasculature stems primarily from evidence that oxidative stress-induced endothelial dysfunction underlies a number of cardiovascular pathologies includ ing hypertension, atherosclerosis, and diabetes, and that antioxidant intervention may be an important treatment modality in these vascular disorders. Of interest is the thiol antioxidant, reduced glutathione (GSH), a crucial regulator of cellular redox potential, and whose synthesis is transcriptionally upregulated under conditions of cellular oxidative stress. The transcriptional upregulation of the rate-limiting enzyme of GSH synthesis, glutamate cysteine ligase (GCL), under oxidative conditions by the transcription factor Nrf2 represents an important area of investigation in terms of its role in redox regulation of endothelial function, its role in vascular pathology, and its potential as a therapeutic target for treatment of cardiovascular disorders that involve vascular oxidative stress. Keywords: GSH redox status and signaling, GSH and vascular function, redox regulation of GSH synthesis, mechanisms of redox signaling. R edox signaling and posttranslational redox modi cations of protein thiols are emerging to be fundamentally important signaling mechanisms in the regulation of mammalian cell function. In addition to redox regulation of cell signaling being a modulator of normal function, a disturbance of redox signaling has also been sug- gested to underpin a variety of pathologies, including vascular diseases. The current chapter will  rst focus on a general discussion of the concept of cellular redox status, the compartmentation of cellular redox systems, and the mechanisms of redox signaling and its targets. The rest of the chapter will be devoted to coverage of the speci c role of vascular-derived reactive oxygen and nitrogen species, the involvement of the glutathione redox system and Nrf2 in the pathways of redox signaling in vascular function and dysfunction, speci c oxidative stress–associated vascular diseases, and antioxidant therapy in treatment of vascular disorders. GENERAL CONSIDERATION OF THE REDOX STATE OF A CELL AND ITS SIGNIFICANCE The redox state of a cell is de ned by the ratio of the interconvertible reduced and oxidized forms of the different cellular redox couples. More generally, the term redox environment has been used to describe the state of the cellular redox pairs (Schafer, Buettner 2001). NOVEL CELLULAR PATHWAYS 474 The intracellular thiol redox pairs are represented by the reduced glutathione/glutathione disul de (GSH/ GSSG), and the reduced and oxidized thioredoxin (Trx/TrxSS) systems, while the cysteine/cystine (Cys/ CySS) redox couple plays an important role in maintaining the redox state of the plasma. The pyridine nucleotide couples include nicotinamide adenine dinucleotide/reduced nicotinamide adenine dinucleotide (NAD + /NADH) and NAD phosphate/reduced NAD phosphate (NADP + /NADPH). The oxidation–reduction status of the redox components is responsible for creating an optimal redox environment within the cell, which directly affects the activity of different cellular proteins. Recently, Hansen et al. proposed that the cellular redox systems are differentially compart- mentalized among different organelles, where the distribution of redox systems is independently controlled in the plasma membrane, cytosol, nucleus, mitochondria, and endoplasmic reticulum (ER) (Hansen, Go, Jones 2006). Thus, depending on the concentrations of the respective redox couples and their  uxes, the compartmentation of speci c redox systems may, in fact, represent a crucial and generalized mechanism for optimizing cell activity within mammalian cells. We have, in recent years established a paradigm that an oxidative shift in the cellular GSH/GSSG redox couple is an important determinant of cell fate; the phenotypic endpoint of proliferation, growth arrest or apoptosis is a function of the extent of GSH/GSSG imbalance (Aw 1999, 2003; Noda, Iwakiri, Fujimoto et al. 2001; Gotoh, Noda, Iwakiri et al. 2002). In various cell types, the loss of GSH/GSSG redox balance preceding cell apoptosis is an early event that occurred within a relatively narrow time window (30 minutes) post–oxidant challenge and is preventable by pre- treatment with the thiol antioxidant, N-acetylcysteine (NAC) (Wang, Gotoh, Jennings et al. 2000; Pias, Aw 2002a, 2002b; Pias, Ekshyyan, Rhoads et al. 2003; Ekshyyan, Aw 2005; Okouchi, Okayama, Aw 2005). These  ndings suggest that GSH/GSSG redox signaling may represent a generalized mechanism in oxidative cell killing in mammalian cells. The control of cellular apoptosis by mucosal GSH/GSSG redox status has been demonstrated in vivo (Tsunada, Iwakiri R, Noda et al. 2003). The current understanding of redox signaling is that it is a regulatory process in which the signal occurs through redox reactions induced by reactive oxygen species (ROS) or reactive nitrogen species (RNS) that results in posttranslational modi cation of proteins in various signal transduction pathways. Many proteins contain cysteine residues that provide a redox-sensitive switch for regulating protein function, and ROS-induced oxidation of cysteine-SH can result in the formation of intra- and/or interchain disul de bonds. Moreover, the direct addition of GSSG leads to S-glutathionylation of the thiol moiety. In addition, nitric oxide (NO • ) can induce S-nitrosylation of speci c cysteine thiols in proteins such as soluble guanylate cyclase (sGC) and the newly discovered mitochondrial NO • /cy tochrome c oxidase signaling pathway (Shiva, Huang, Grubina et al. 2007; Landar, Darley-Usmar 2007). These redox signal transduction processes are important in various physiological and biological activities including vascular function. CONCEPT OF OXIDATIVE AND NITROSATIVE STRESS AND REDOX SIGNALING In redox signaling, modi cations of targeted proteins are initiated by ROS and RNS. The common ROS are superoxide anions (O 2 •– ), hydrogen peroxide (H 2 O 2 ), and hydroxyl radicals (HO•) while RNS comprise NO • and its derivatives, peroxynitrite (ONOO – ) or dinitrogen trioxide (N 2 O 3 ) (Fig. 19.1). Endogenous sources of O 2 •– include the mitochondrial respiratory chain, NADPH oxidase, xantine oxidase, and NADPH cytochrome P450 (Cross, Jones 1991). Intracellular derived O 2 •– is readily dismutated to H 2 O 2 by cytosolic or mitochondrial superoxide dismutases (SOD). In the presence of metal ions, H 2 O 2 and O 2 •– are con- verted to HO•, a highly potent oxidant that induces oxidative damage to cellular proteins, lipids, or DNA. Exogenous sources such as xenobiotics or UV/γ- radiations are known ROS generators that contribute to the overall oxidant burden of a cell. O 2 •– can further react with NO • to form the reactive ONOO – that oxidizes cellular lipids or DNA, resulting in nitrosative stress (for review see Pacher, Beckman, Liaudet 2007). NO • is generated by NO synthases (NOS), of which three isoforms exist in mammalian cells; these are endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). In the vasculature, eNOS is the predominant isoform and is responsible for maintaining vascular NO • homeostasis and vascular tone. Oxidation of the essential NOS cofactor by ONOO – transforms eNOS into an ROS producer (Forstermann, Closs, Pollock et al. 1994). Another RNS derivative with a potential role in cellular signaling is N 2 O 3 , which participates in the nitrosation of thiol groups to form nitrosothiols, an important class of redox signaling molecules. Endothelial ROS and RNS generation and their speci c roles in vascular function are discussed in sections on cellular sources of endothelial ROS and ROS and vascular signaling. It is well recognized that different concentrations of ROS/RNS mediate distinct cellular responses. While high ROS/RNS concentration induces oxidative damage to macromolecules that lead to oxidative/ Chapter 19: Redox Signaling and Vascular Function 475 COMPARTMENTATION OF CELLULAR REDOX SYSTEMS AND REDOX PROTEINS IN CELL SIGNALING GSH/GSSG Redox System and its Subcellular Compartmentation Given its cellular abundance and its role in protein thiol modi cation, the status of the GSH/GSSG redox system re ects the redox buffering capacity of a cell. Indeed, an oxidative shift in the GSH-to-GSSG ratio is often used as an indicator of cellular oxidative stress. GSH/GSSG and Cellular Redox Balance GSH (γ-glutamylcysteinyl glycine) is the most abundant low-molecular-weight thiol in cells that exhibits important roles in the control of the thiol–disul de redox state of cellular proteins (Meister, Anderson 1983; Sies 1999). Additionally, GSH is involved in redox activation of transcription factors as part of an adaptive mechanism that participates in cell signaling and stress responses (Kamata, Hirata 1999). Within cells, GSH exists mainly in its biologically active, thiol-reduced form, and oxidation of GSH results in the formation of glutathione disul de, GSSG. The ratio of GSH and GSSG is maintained in favor of the nitrosative stress, low-to-moderate levels are important in cell signaling and the regulation of various biological processes. The balance of detrimental and bene cial actions caused by ROS/RNS is achieved through “redox regulation” mechanisms; this refers to enzymatic reactions with speci c roles in maintaining the redox homeostasis of targeted proteins that are essential for cell function and survival (Droge 2002). The cytotoxic effects of ROS/RNS are amelio- rated by intracellular antioxidant mechanisms that maintain a balance of the reduced and oxidized species (Fig. 19.1). For example, H 2 O 2 and hydroperoxides are eliminated by GSH peroxidase at the expense of cellular GSH, and the resultant increase in GSSG is reduced by glutathione reductase with NADPH as the reductant. Peroxiredoxin (Prx) is another example of antioxidant redox proteins that are involved in the breakdown of cellular toxic hydroperoxides. Previous studies have demonstrated that these cysteine-speci c peroxidases can function as molecular switches sensitive to different levels of H 2 O 2 (Bozonet, Findlay, Day et al. 2005). The Trx/Trx reductase and glutaredoxin (Grx)/GSH system also contribute to cellular antioxidant defense against redox imbalance as do the sul redoxins (Srx) in the reduction of oxidized proteins (Fig. 19.1; section on redox proteins and cell signaling). Figure 19.1 Metabolic pathways of ROS/RNS formation and the interactions with antioxidant systems. ROS are formed from exogenous and endogenous sources. While highly reactive HO• induces oxidative stress, O2 •– and H 2 O 2 are either substrates for antioxidant enzymes or induce sequential oxidation of Cys residues of target proteins of various signaling pathways. The formation of GSSG can participate in the S-glutathionylation of proteins (P-SSG). NO radicals produced by NOS isoenzymes can form diverse RNS intermediates that either participate in S-nitrosylation of Cys residues of proteins to form GSNO/P-SNO derivatives or induce nitrosative stress and the formation of tyrosine nitrosative derivatives. The redox status of proteins is restored by the GSH, Trx, Grx, and the newly discovered Srx redox systems. CAT, catalase; GSH, glutathione; GPx, GSH peroxidase; GR, GSH reductase; Grx, glutaredoxin; GSSG, glutathione disulfi de; NO • nitric oxide radical; NOS, nitric oxide synthases; N 2 O 3 , dinitrogen trioxide; ONOO – , peroxynitrite; SOD, superoxide dismutase; Srx, sulfi redoxin; Trx, thioredoxin. Exo- and endogenous sources NOS ONOO - GSH P-SH GSNO GSH GSH P-SH P-SNO P-SSG P-SH Trx, Grx, GSH, Srx Cys-oxidation (P-SO n H) Nitrosative stress NO SOD CAT GSH H 2 O 2 HO• GSSG GPx GR NADP + Fe 3+ Fe 2+ NADPH Glucose O 2 + H 2 O N 2 O 3 Oxidative stress Cys-oxidation (P-SO n H) O 2 •– NOVEL CELLULAR PATHWAYS 476 in excess of 100-to-1 in liver cells, and this ratio signi - cantly decreases to less than 4 to 1 during oxidative stress. The mitochondria maintain a distinct GSH pool that is supported through GSH transport from the cytosolic compartment via the dicarboxylate and 2-oxoglutarate GSH carriers located in the mitochondrial inner membrane (Chen, Lash 1998). This GSH redox compartment is metabolically separate from the cytosol with regard to synthetic rate, turnover, and sensitivity to chemical depletion. Matrix GSH concentrations are between 5 and 10 mM and varies from 10% to 15% of the total GSH in the liver (Jocelyn, Kamminga 1974) to 15% to 30% of total GSH pool in the renal proximal tubule (Schnellmann 1991). Functionally, mitochondrial GSH preserves the integ- rity of mitochondrial proteins and lipids and controls mitochondrial generation of ROS. Early studies demonstrated that the status of mitochondrial GSH is a determining factor in oxidative vulnerability; in this regard, mitochondrial GSH loss has been linked to cytotoxicity induced by aromatic hydrocarbons (Hallberg, Rydstrom 1989), hypoxia (Lluis, Morales, Blasco et al. 2005), tert- butylhydroperoxide (tBH) (Olafsdottir, Reed 1988), and ethanol intoxication reduced state (>90% reduced), which is accomplished by three mechanisms: GSH synthesis, GSSG reduction, and GSH uptake. De novo synthesis of GSH from precursor amino acids (glutamate, cysteine, and glycine) occurs in the cytosol and is catalyzed by two ATP- dependent enzymatic reactions, γ-glutamate cysteine ligase (GCL) and GSH synthetase (GS) (Fig. 19.2). GCL activity is rate limiting in GSH synthesis and is regulated by GSH and the availability of cysteine. GSSG reduction is catalyzed by glutathione reductase, and uptake of extracellular GSH occurs through speci c carriers localized at the plasma membrane (Lash, Putt, Xu et al. 2007). Intracellular Compartmentation of GSH In mammalian cells, GSH is present in millimolar concentrations and is differentially distributed among various cellular compartments, such as the cytosol, mitochondria, ER, and nucleus where it forms separate and distinct redox pools (Fig. 19.2). Within the cytosol, GSH concentrations are maintained between 5 mM and 10 mM (Meister, Anderson 1983) and the redox pool is highly reduced; for example, the GSH- to-GSSG ratio under normal conditions is maintained Figure 19.2 GSH synthesis and compartmentation of thiol/disulfi de redox state and redox proteins. Synthesis of GSH from its constituent amino acids (glutamate, cysteine, glycine) takes place in the cytosol and is catalyzed by glutamate cysteine ligase (GCL) and glutathione synthase (GS) at the expense of two moles of ATP. Some cells can export GSH or Trx1 which, once outside the cells, can contribute to maintaining the redox environment of the plasma. Extracellular GSH is hydrolyzed to its component amino acids by γ-glutamyltransferase (γ-GT) and dipeptidase (DP). The main redox couples that participate in maintaining the cellular reduced-to-oxidized environment include GSH/GSSG and Trx/TrxSS. The cysteine residues in cellular proteins are maintained in the reduced state by GSH and thiol reductases, namely, Trx, Grx, Nrx, or PDI that have different cellular localization in the cytoplasm, mitochondria, endoplasmic reticulum, and nucleus. Cys, cysteine; Grx, glutaredoxin; GSH, glutathione; GSSG, glutathione disulfi de; Nrx, nucleoredoxin; PDI, protein disulfi de isomerase; PrSH, reduced protein; Pr-SSG, oxidized protein; Trx, thioredoxin; TrxSS, oxidized thioredoxin; Trx80, truncated form of Trx. Cytoplasm GSH (5–10 mM) Trx1 (2–14 µM) Grx1 Trx1 or Trx80 GSH/GSSG Trx1/TrxSS GSH/GSSG GSH/GSSG GSH/GSSG Prot-SH/ Prot-SSG GSH PDI GSH Trx1 Grx2 Nrx Trx1/TrxSS Trx2/TrxSS GSH/GSSG GSH Trx2 Grx2 Glu + Cys g-glu-cys gGT Cys, Gly DP GS GSH Nucleus Endoplasmic reticulum Mitochondria ATP ADP Extracellular GSH ATP ADP GCL Chapter 19: Redox Signaling and Vascular Function 477 current evidence suggests a passive diffusion of GSH from the cytosol into the nucleus via nuclear pores (Ho, Guenthner 1997). Redox Proteins and Cell Signaling Members of the Trx family of proteins are active in the redox regulation of cysteine residues of special- ized proteins that signi cantly impact cell signaling and function. Among the better-studied redox proteins are Trx, Grx, peroxiredoxin, and protein disul-  de isomerase (PDI). Thioredoxins Thioredoxins (Trx) are small ubiquitous redox proteins that contain two redox-active cysteine residues in the catalytic site (Cys-X-X-Cys). The intracellular concentrations of Trx are between 2 µM and 14 µM, which is three orders of magnitude lower than that of GSH (2 mM to 10 mM) (Nakamura, Nakamura, Yodoi 1997). Trx, together with NADPH and Trx reductase, functions to maintain the thiol/disul de redox state of proteins in mammalian cells. Trx catalyzes the reversible reduction of disul de bonds in oxidized proteins at the expense of cysteine residues in its active motif site; the active reduced Trx is regenerated by Trx reductase and NADPH. Mammalian cells contain two forms of Trx, Trx1, and Trx2, which are localized in different cellular compartments, cytosol, mitochondria, or nucleus. Trx 1 is expressed ubiquitously and is a cytosolic enzyme that can translocate to the nucleus during oxidative stress. Cytosolic Trx1 can function as a cofactor, binding partner, or reductant. For example, as a cofactor for Prx, Trx1 functions in hydroperoxide elimina- tion. The binding of Trx1 with the apoptosis signal– regulated kinase 1 (ASK-1) plays an anti-apoptotic role (Saitoh, Nishitoh, Fujii et al. 1998). As a reductant, Trx1 and Trx-like protein (TRP14) reactivate the protein tyrosine phosphatase (PTP), phosphatase- like tensin homolog (PTEN), which reverses phos- phoinositide 3-kinase (PI3K) signaling (Lee, Yang, Kwon et al. 2002). Through reduction of protein disul des, Trx functions in redox-sensitive signaling and the activation of transcriptional factors (for review see Watson, Yang, Choi et al. 2004). During oxidative challenge, Trx1 translocates to the nucleus where it participates in the redox regulation of transcription factors such as activator protein 1 (AP-1), p53, and nuclear transcription factor kappa B (NF-κB). Trx1 involvement in redox control of transcriptional activity is supported by the observation that the redox state of nuclear Trx1 is more reduced than that of the cytosolic protein (Watson, Jones 2003). Several studies (Fernandez-Checa, Garcia-Ruiz, Ookhtens et al. 1991). Our recent studies validated that oxidant- induced apoptosis is triggered by a loss of mitochondrial GSH/GSSG balance (Circu, Rodriguez, Maloney et al. 2008). Mechanistically, oxidative susceptibility is associated with an increase in mitochondrial ROS production secondary to matrix GSH decrease (Lluis, Buricchi, Chiarugi et al. 2007). The existence of a distinct GSH pool in the ER has been described and its concentration (6 mM to 10 mM) mirrors those in the cytosolic and mitochondrial compartments (Fig. 19.2). Notably, the GSH redox environment in ER is highly oxidized (GSH-to- GSSG ratio of 3:1–1:1), a state that favors the oxidative folding of proteins (Bass, Ruddock, Klappa et al. 2004). The luminal GSSG concentration is, indeed, optimal for disul de bond formation (Lyles, Gilbert 1991), and appears to be generated through an oxidative pathway catalyzed by the oxidoreductase enzyme, Er01 (Tu, Weissman 2004). Also notable is that less than 50% of the ER thiol pool (GSH + GSSG) is free; the majority of GSH is reversibly bound to proteins as protein-mixed disul des formed through thiol oxidation by GSSG. Functionally, it is believed that high concentrations of protein-mixed disul des serve as GSH reserve within the ER, in the maintenance of oxidoreductase catalytic function, or as a redox buf- fer against ER-generated ROS (Jessop, Bulleid 2004; Chakravarthi, Jessop, Bulleid 2006). Elevated levels of reduced GSH disrupt ER function and activate the unfolded protein response (UPR) that triggers cellular apoptosis (Frand, Kaiser 2000). An independent nuclear GSH pool functions in DNA synthesis and protection against oxidative and ionizing radiation induced DNA damage (Cotgreave 2003). The size of the nuclear GSH pool is unknown, and recent evidence suggests that the cytosolic and nuclear redox pools are not in equilibrium. Bellomo and coworkers (Bellomo, Palladini, Vairetti 1997) demonstrated a GSH ratio of 3:1 between the nuclear and cytosolic compartments, while Thomas et al. (1995) and Soderdahl et al. (2003) reported lower ratios. Moreover, nuclear proteins are more prone to thiol oxidation (Soderdahl, Enoksson, Lundberg et al. 2003). Interestingly, nuclear GSH distribution is dynamic and directly correlates with cell cycle progression where nuclear GSH was 4-fold higher than cytosolic GSH in the proliferative state, but was equally distributed between the two compartments when cells reached con uency (Chen, Delannoy, Odwin et al. 2003; Markovic, Borras, Ortega et al. 2007). These results suggest a speci c role for nuclear GSH in pre- serving nuclear proteins in a reducing environment that is essential for gene transcription during cell cycle progression (Chen, Delannoy, Odwin et al. 2003). The mechanism for nuclear GSH transport is unresolved; [...]... binding of AP-1 is attenuated by S-glutathionylation of the cysteine residue in the AP-1 catalytic site in response to decreased cellular GSH/GSSG ratio (Klatt, Molina, De Lacoba et al 199 9) Similarly, NO• modulates AP-1 DNAbinding by reversible S-nitrosylation (Cys272 of c-Jun), but this modification is cell-type specific (Morris 199 5; Klatt, Molina, Lamas 199 9) Indirectly, ROS stimulates AP-1 activity... activation/↑NF-κB and AP-1 Vascular complication of diabetes Higai et al 2006 ↑NADPH oxidase/↑ROS/JNK1 and p38 MAPK activation/↑ c-Fos, c-Jun and JunB expression, ↑AP-1 activity Mitogenesis associated with atherosclerosis, aging, or cancer Rao et al 199 9 ↑ROS-RNS/↑AP-1 activity/↑MMP-2/cardiac remodeling Response to I/R injury Alfonso-Jaume et al 2006 ↑I/R-ROS/NF-κB and AP-1 activation/ICAM-1 upregulation/acute... 199 5 (Mulcahy, Gipp 199 5), the human GCLc promoter notably contained consensus sites for AP-1, AP-2, SP-1, and Nrf2 Four AREs are uncovered (Mulcahy, Wartman, Bailey et al 199 7), of which the most distal ARE4 is responsible for constitutive and β-naphthflavone (β-NF)–induced GCLc promoter activity; specifically, a TRE within ARE4 controls constitutive GCLc promoter activity (Wild, Gipp, Mulcahy 199 8)... and NO signaling functions Cell 102(4): 499 –5 09 Fachinger G, Deutsch U, Risau W 199 9 Functional interaction of vascular endothelial-protein-tyrosine phosphatase with the angiopoietin receptor Tie-2 Oncogene 18(43): 594 8– 595 3 Fan H, Sun B, Gu Q, Lafond-Walker A, Cao S, Becker LC 2002 Oxygen radicals trigger activation of NF-kappaB and AP-1 and upregulation of ICAM-1 in reperfused canine heart Am J Physiol... a +2 state) and sulfonic acid (–SO3H, a + 4 state) are 480 NOVEL CELLULAR PATHWAYS P S P P-SH S P-SH P-SOH GSH GSSG ROS (Disulfide bond formation) (Cys P-SO3H P-SO2H oxidation) Irreversible modifications P-SSG GSH (S-glutathionylation) GSNO RNS P-SH P-SNO (S-nitrosylation) Figure 19. 3 Redox modification of protein thiols with role in cellular signaling Cysteine residues from target proteins can undergo... on activation, ASK-1 signaling leads to the activation of p38 and JNK pathways (Nishitoh, Saitoh, Mochida et al 199 8) Chapter 19: Redox Signaling and Vascular Function ROS Trx1 ASK-1 Trx-ASK-1 redox sensor Trx1 ASK-1 TrxSS TRAF2/6 Activated ASK-1 signalosome ROS P ASK-1 TRAF2/6 ASK-1 TRAF2/6 Mitochondria Trx2 Mito ASK1 P TRAF2/6 MKKs JNK Cyt c Apoptosis↑ Figure 19. 4 ROS-mediated ASK-1 signaling and... necrosis factor-α, ionizing radiation, and ROS leads to NF-κB activation (Baeuerle 199 8) It is well recognized that NF-κB activation and DNA binding are sensitive to the cellular redox status For example, NF-κB/DNA binding is decreased by thiol oxidants such as diamide and increased by thiol-reducing compounds such as dithiothreitol and β-mercaptoethanol (Hayashi, Ueno, Okamoto 199 3) Cytosolic NF-κkB activation... dissociation/↑Nrf2/ARE-responsive genes Vascular proliferation Villacorta et al 2007 EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HO-1, heme oxygenase 1; JNK, c-Jun N-terminal kinase; Keap1, Kelch-like ECH-associated protein 1; LNO2, nitro-linoleic acids; moxLDL, moderately oxidized LDL; MAPK, mitogen-activated protein kinase; NO, nitric oxide Table 19. 2 Cellular Pathways... inhibition of NF-κB activation (Hirota, Murata, Sachi et al 199 9) whereas within the nucleus, Trx1 reduces Cys62 of the p50 subunit of NF-κB, resulting in increased binding to DNA (Matthews, Wakasugi, Virelizier et al 199 2) S-glutathionylation is another mode of redox control of NF-κB activity S-glutathionylation of Cys62 of the p50 subunit has been shown to inhibit NF-κB binding to DNA (Pineda-Molina, Klatt,... endothelial surface expression of adhesion molecules (E-selectin, Chapter 19: Redox Signaling and Vascular Function ICAM-1 and VCAM-1) serve to tether circulating leukocytes to the endothelium which increased O2•– and H2O2 formation For instance, endothelial VCAM-1 and ICAM-1 cross-linking respectively stimulates Nox-dependent (Matheny, Deem, Cook-Mills 2000) and XO-dependent O2•– production (section VII.1) Additional . al. 199 9). Similarly, NO • modulates AP-1 DNA- binding by reversible S-nitrosylation (Cys 272 of c-Jun), but this modi cation is cell-type speci c (Morris 199 5; Klatt, Molina, Lamas 199 9) S-glutathionylation. In addition, GSNO can induce both S-nitrosylation and S-glutathionylation of protein thiols. P-SH P-SH P-SOH GSH GSSG ROS RNS GSH GSNO P-SNO P-SSG P-SO 2 H P-SO 3 H P-SH PS PS Irreversible modifications (Disulfide. Srx, sulfi redoxin; Trx, thioredoxin. Exo- and endogenous sources NOS ONOO - GSH P-SH GSNO GSH GSH P-SH P-SNO P-SSG P-SH Trx, Grx, GSH, Srx Cys-oxidation (P-SO n H) Nitrosative stress NO SOD CAT GSH H 2 O 2 HO• GSSG GPx GR NADP + Fe 3+ Fe 2+ NADPH Glucose O 2 +