Endogenous Reactive Intermediates as Modulators of Cell Signaling

FEBRUARY 2006 VOLUME 19, NUMBER 2 © Copyright 2006 by the American Chemical Society

ReViews Endogenous Reactive Intermediates as Modulators of Cell Signaling and Cell Death James D. West† and Lawrence J. Marnett* Department of Biochemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt-Ingram ComprehensiVe Cancer Center, and Center in Molecular Toxicology, Vanderbilt UniVersity School of Medicine, 23rd AVenue at Pierce, NashVille, Tennessee 37212-0146 ReceiVed NoVember 16, 2005

1. Introduction 2. Oxidative Injury: Reactive Oxygen Species (ROS) Generation and Downstream Consequences 2.1. Generation of ROS 2.2. DNA and Protein Damage by ROS 2.3. Lipid Peroxidation 2.4. Aldehydic Products of Lipid Peroxidation 2.4.1. Macromolecular Targets of R,β-Unsaturated Aldehydes 2.4.2. Detoxification 2.4.3. Evaluation of in Vivo Production 3. Cell Death Mechanisms Activated by Endogenous Reactive Intermediates 3.1. Necrosis 3.2. Apoptosis 3.2.1. Caspases 3.2.2. Mechanisms of Caspase Activation 3.2.3. Caspase Targets 4. Cell Signaling Pathways Influenced by Reactive Intermediates Which Alter Cell Death 4.1. Antioxidant Response Signaling 4.2. Heat Shock Response Signaling

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* To whom correspondence should be addressed. Tel: 615-343-7329. Fax: 615-343-7534. E-mail: [email protected]. † Present address: Department of Biochemistry, Molecular Biology, and Cell Biology, Rice Institute for Biomedical Research, Hogan 2-100, 2205 Tech Drive, Northwestern University, Evanston, IL 60208-3500.

4.3. Nutrient Deprivation and ER Stress Response Signaling 4.4. Stress Responsive MAP Kinase Signaling 4.5. NF-κB Signaling 4.6. DNA Damage Response Signaling 5. Complexity in Oxidative Stress Signaling: Lessons Learned from HNE 5.1. Cellular Responses Induced by HNE 5.2. Macromolecular Targets of HNE 6. The Future

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1. Introduction One of the most active areas of biological investigation in the past two decades has been the study of cell death mechanisms. Cell death occurs during numerous biological processes, ranging from beneficial effects in neurological development, autoreactive immune cell elimination, and precancerous cell clearance to adverse effects in chronic inflammation, neurodegeneration, diabetes, and cardiovascular disease. The modes of cell death exhibit a broad continuum of cytological and biochemical changes, ranging from accidental cell rupture (termed necrosis) to the events of programmed cell death (termed apoptosis) that are regulated by a family of cysteine proteases called caspases (1). The agents that induce such cell death responses are equally diverse and include death-inducing proteins [e.g., Fas ligand (FasL),1 tumor necrosis factor R (TNFR)], a variety of clinically used drugs (e.g., DNA-targeted chemotherapies, proteasome inhibitors), several forms of envi-

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174 Chem. Res. Toxicol., Vol. 19, No. 2, 2006

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Scheme 1. Generation of ROS and Reactive Nitrogen Species

ronmental injury (e.g., radiation exposure, heat shock), and reactive intermediates (e.g., H2O2, peroxynitrite, and lipid peroxidation products). Here, we review the production of endogenous reactive intermediates, the mechanistic features of cell death, and the signaling pathways that these intermediates ultimately alter to influence cell death processes.

2. Oxidative Injury: Reactive Oxygen Species (ROS) Generation and Downstream Consequences ROS represent a major class of endogenous cell signaling agents and cytotoxic effectors that are believed to contribute to numerous diseases including aging (2-4). A prevailing theory for the progression of aging is that accumulation of ROS and associated damage throughout life brings about cell death (5, 6). As a consequence, research on aging and age-related disorders has focused intensely on understanding the production of reactive oxygen intermediates and modification of their downstream targets as a potential route to degenerative cell death.

2.1. Generation of ROS ROS production is largely the result of living in an aerobic environment. Superoxide (O2-) arises during aberrant mitochondrial respiration through reduction of molecular oxygen by semiubiquinone (Scheme 1) (7). It is estimated from in vitro experiments that 1-2% of O2 consumed by mitochondria is converted to O2- (6). O2- also is produced through various enzymatic oxidation reactions (e.g., reactions catalyzed by cytochromes P450 and other oxidoreductases) (6, 7). During the innate immune response, O2- is also generated by activation of NADPH oxidase. This processsthe respiratory burstsoccurs to ward off harmful pathogens (6, 7). Once produced, O2- reacts 1Abbreviations: ROS, reactive oxygen species; MAPK, mitogenactivated protein kinase; HNE, 4-hydroxy-2-nonenal; FasL, Fas ligand; TNFR, tumor necrosis factor R; SOD, superoxide dismutase; MPx, myeloperoxidase; GPx, GSH peroxidase; 13-HPODE, 13-hydroperoxy-9,11-octadecadienoic acid; HPNE, 4-hydroperoxy-2-nonenal; ONE, 4-oxo2-nonenal; FDP-lysine, N-(3-formyl-3,4-dehydropiperidino) lysine; GST, GSH transferase; ALDH, aldehyde deydrogenase; AKR, aldo-keto reductase; ER, endoplasmic reticulum; PARP-1, poly(ADP-ribose) polymerase-1; ICE, interleukin 1-β-converting enzyme; Smac/DIABLO, second mitochondrial activator of caspases/direct IAP binding protein with low pI; Bcl-2, B cell lymphoma-2; Apaf-1, apoptotic protease activating factor-1; IAP, inhibitor of apoptosis protein; DFF, DNA fragmentation factor; HSF1, heat shock factor 1; Hsp, heat shock protein; Grp78/BiP, glucose regulated protein 78/immunoglobulin binding protein; ATF, activating transcription factor; XBP1, X-box binding protein 1; JNK, c-Jun N-terminal kinase; MKP, MAPK phosphatase; IKK, IκB kinase; Trx, thioredoxin; TrxR, thioredoxin reductase.

Figure 1. Structures of oxidized DNA bases and amino acid side chains. Oxidation of nucleobases in DNA (A) and amino acids in proteins (B) leads to many products; several are shown.

at diffusion-controlled rates with NO• (a product of nitric oxide synthases) to produce a diverse panel of oxidizing and nitrosating/nitrating species (8, 9), or it rapidly dismutates nonenzymatically or enzymatically with the aid of superoxide dismutases (SODs) to O2 and hydrogen peroxide (H2O2) (10). H2O2 is utilized by myeloperoxidase (MPx) in inflammatory cells to produce hypochlorous acid and other noxious chlorinederived oxidants (11). Additionally, H2O2 can be reduced to hydroxyl radical (•OH) through the Fenten reaction, wherein Fe2+ or Cu2+ functions as a reducing agent (12). Acting as a powerful oxidant, •OH can damage a number of biological macromolecules, even though it has a very short lifetime (7). It is estimated that •OH only diffuses within five molecular diameters before it oxidizes a target (7). To avoid the formation of •OH, H2O2 is reduced to H2O by either GSH peroxidase (GPx) or catalase (12). If these detoxification systems are compromised or if ROS production is excessive, oxidative stress results, and DNA, proteins, and lipids are subject to damage (13). As a result, ROS represent a major class of biologically important cytotoxic molecules, capable of damaging proteins, DNA, and lipids directly to influence cell signaling and cell fate decisions.

2.2. DNA and Protein Damage by ROS Once produced, some species of ROS can oxidize virtually any biological macromolecule. The fact that there are numerous cellular mechanisms in existence to limit accumulation of ROS (e.g., SOD, GPx, and catalase) suggests that pronounced damage of downstream targets is undesirable (14). Oxidative damage of DNA results in the formation of several premutagenic lesions, commonly occurring as 8-oxo-deoxyguanosine, 8-oxo-deoxyadenosine, and deoxythymidine glycol, among others (Figure 1A) (13). Many of these oxidized DNA bases are formed in vivo, are selectively excised from DNA by DNA glycosylases, and are mutagenic in various model systems (13, 15-17).

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Although the existence of oxidized DNA bases in vivo is unquestioned, whether such forms of DNA damage represent reliable biomarkers of oxidative injury is a contentious issue, as the extent of oxidative damage can be overestimated and is highly variable (18). Oxygen radicals also damage the peptide backbone and individual amino acids in proteins. Either type of modification may have pronounced functional effects on proteins. Hydrogen atoms are abstracted from the R-carbon in peptide chains or from side chains of aromatic residues, and the resulting radicals are trapped by O2 (19, 20). Sulfur atoms in cysteine and methionine are common targets for oxidation as well, and their modification can have functional consequences (Figure 1B) (19). Many irreparable forms of oxidative protein damage (i.e., protein carbonyls resulting from breakdown of the peptide backbone, oxidized aromatic residues) are reliable indicators of oxidative injury in vivo (21, 22), as levels of oxidized proteins and/or amino acids increase with aging and are elevated in diseased tissues where oxidative stress has been implicated.

Scheme 2. Lipid Peroxidation

2.3. Lipid Peroxidation Polyunsaturated fatty acids in membranes represent major targets of intracellular oxidizing agents. Because most ROS arise in or around membrane-rich organelles (23), it is understandable why the process of lipid peroxidation has received considerable attention as a branch of oxidative stress biology. Many of the products of lipid oxidation are chemically reactive, inducing alterations in cell signaling and survival through their ability to modify various cellular targets. Lipid peroxidation occurs as a series of radical chain reactions and is initiated by radical-mediated abstraction of a bis-allylic hydrogen atom from either the polyunsaturated ω-3 or ω-6 fatty acids (Scheme 2) (24-26). Polyunsaturated fatty acids (e.g., linoleic acid, arachidonic acid, linolenic acid, and docosahexaenoic acid) are constituents of glycerophospholipids and other lipids in plasma and organelle membranes (27). Their oxidation in membranes may be carried out by several oxidants, including HO• arising from the Fenten reaction (27, 28). Following hydrogen abstraction, the delocalized radical reacts with O2 through radical coupling and forms a lipid peroxyl radical (LOO•) (25, 26). Subsequently, a chain reaction is propagated by LOO•, abstracting a hydrogen atom from a polyunsaturated lipid to generate L• (25, 26). During the propagation phase, many molecules of polyunsaturated fatty acids become damaged. Lipid peroxidation through nonenzymatic means generates a number of lipid hydroperoxides, which themselves undergo a variety of chemical changes. LOO• can cyclize to form endoperoxides. Bicyclic endoperoxides often decompose nonenzymatically to yield malondialdehyde, which exists predominantly as β-hydroxyacrolein under normal conditions (27, 29). Alternatively, some lipid hydroperoxide molecules undergo a second round of oxygenation by hydrogen abstraction at different sites of unsaturation. These doubly oxidized lipids can decompose to yield 4-hydroperoxy2-nonenal (HPNE) and a family of related, long chain R,βunsaturated aldehydes (Scheme 3) (28, 30, 31). Additionally, a number of other short chain, R,β-unsaturated aldehydes (e.g., acrolein, crotonaldehyde) are produced during lipid peroxidation through poorly understood mechanisms (Figure 2) (31-35). Acrolein is also derived via the MPx-catalyzed oxidation of the amino acid Thr (36). Many of the decomposition products of oxidized polyunsaturated fatty acids are highly reactive and exhibit their own spectrum of damaging effects, similar to those of ROS (13, 27, 37, 38).

Scheme 3. Derivation of HNE and Related Nonenals from 13-HPODE

In recent years, the decomposition of 13-HPODE (13hydroperoxy-9,11-octadecadienoic acid, an autoxidation product of linoleic acid) has received much attention as a potential biological source of 4-hydroxy-2-nonenal (HNE) (Scheme 3). In these studies, two other closely related aldehydes have been identified (28, 30, 39). HPNE is the proposed immediate


Figure 2. Structures of some aldehydic products of lipid peroxidation. Although MDA, HPNE, HNE, and ONE represent major products of lipid peroxidation in vitro, other molecules produced through alternative or unknown mechanisms include acrolein, crotonaldehyde, 4-hydroxyhexenal, and 9,12-oxo-10-dodecenoic acid. Each of these aldehydic products can produce its own damaging effects.

degradation product of 13-HPODE, and it can dehydrate to 4-oxo-2-nonenal (ONE) or be reduced to HNE (28, 30). A unique aspect of the production of HPNE, ONE, and HNE from 13-HPODE is that this reaction can be enhanced considerably in vitro by incubation with ascorbic acid (28, 35). Additionally, ascorbic acid may play a direct role in scavenging these molecules (35).

2.4. Aldehydic Products of Lipid Peroxidation Aldehydes generated during the lipid peroxidation cascade exhibit a wide range of biological effects, ranging from DNA damage and mutagenesis to signaling pathway alteration. These effects are indeed similar to the detrimental effects caused with ROS accumulation, demonstrating the complexity inherent in studying oxidative injury. The macromolecular targets and detoxification of R,β-unsaturated aldehydes are discussed below, as understanding the chemical and biochemical nature of these molecules is essential in elucidating their signaling properties.

2.4.1. Macromolecular Targets of r,β-Unsaturated Aldehydes Most R,β-unsaturated aldehydes exhibit varying degrees of reactivity with individual nucleotides within nucleic acids.

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Guanine is the most commonly modified DNA base because of its high nucleophilicity (Scheme 4A and Figure 3A) (13). However, reaction of R,β-unsaturated aldehydes with cytosine and adenine has also been observed (16). The products are typically six-membered ring, exocyclic dG adducts. Several exocyclic adducts derived from lipid peroxidation products undergo conversion to the ring-opened aldehyde form when paired opposite deoxycytidine in DNA (40, 41), which generates an aldehyde that can form Schiff base adducts with primary amines (42-45). The net result of DNA damage generated by R,β-unsaturated aldehydes is an increased propensity for mutation, commonly in the form of G-to-T transversions and frameshifts (45-47). Proteins also represent major targets of R,β-unsaturated aldehydes, causing many of the signaling effects discussed in the latter part of this review. Nucleophilic amino acids (i.e., Cys, His, and Lys) are most commonly modified by R,βunsaturated aldehydes (Scheme 4B). In the case of MDA and acrolein, Lys residues in target proteins are the most susceptible to modification, causing the formation of lysinepropenal and N-(3-formyl-3,4-dehydropiperidino-) (FDP-) lysine, respectively (Figure 3B) (27, 32, 33). HNE and closely related molecules form Michael adducts with Cys, His, and Lys residues, which, in the case of HNE, rearrange to form more stable hemiacetals (Figure 3B) (27, 48-51). Additionally, HNE can form Schiff base adducts that dehydrate to 2-pentylpyrrole adducts or rearrange to form fluorescent species (Figure 3B) (52-54). Because of their bifunctional nature, many of these aldehydes are capable of forming protein-protein cross-links and potentially stimulating higher order protein aggregation (49, 53, 55).

2.4.2. Detoxification Most highly reactive R,β-unsaturated aldehydes are inactivated in multiple ways, including conjugation to GSH, oxidation by aldehyde dehydrogenases, or reduction by aldoketoreductases. Strongly electrophilic lipid peroxidation products induce rapid and substantial depletion of intracellular GSH, suggesting that this is perhaps the most important detoxification mechanism (27). Once GSH is depleted, cells exhibit an intracellular change

Scheme 4. DNA and Protein Damage Resulting from Aldehydic Products of Lipid Peroxidation

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Figure 3. Structures of DNA bases and amino acids modified by R,β-unsaturated aldehydes. Shown are examples of exocyclic deoxyguanosine adducts (A) and modified amino acid side chains in proteins (B) generated by R,β-unsaturated aldehydes.

in redox status (56). It is likely that these aldehydes propagate an oxidative stress response following their production (37). Reaction of R,β-unsaturated aldehydes with GSH can proceed in one of two waysseither by nonenzymatic conjugation or through GSH transferase (GST)-mediated conjugation to form Michael adducts. GSTA4-4 is the principle GST responsible for metabolism of most long chain R,β-unsaturated aldehydes, although many GSTs may be involved in the metabolism of these molecules (57, 58). Several oxidoreductases also inactivate HNE and related aldehydes. Particularly, detoxification of HNE in this manner is accomplished by oxidation or reduction of the aldehyde or by reduction of the 2,3-double bond. Aldehyde dehydrogenase 3 (ALDH3) is responsible for the conversion of HNE to 4-hydroxy-2-nonenoic acid, a noncytotoxic molecule (59). Aldose reductase and aldo-keto reductase AKR1C1 efficiently reduce HNE to 1,4-dihydroxy-2-nonene (58, 60). Reduction of the 2,3-double bond of HNE is carried out by 15-oxoprostaglandin oxidoreductase, forming 4-hydroxynonanal (61). Recent reports suggest that related molecules (e.g., ONE) are metabolized in a similar fashion (62, 63). If these metabolic pathways are compromised or overcome by excessive aldehyde accumulation, reaction with DNA and proteins occurs, potentially leading to adverse alterations in cellular homeostasis.

2.4.3. Evaluation of in Vivo Production To date, most studies examining the production of R,βunsaturated aldehydes produced during oxidative stress in vivo

have been qualitative in nature. This is largely due to technical difficulties associated with directly measuring steady state levels of R,β-unsaturated aldehydes, as they exhibit high reactivity with cellular targets. Nonetheless, several studies have linked R,βunsaturated aldehyde production with oxidative injury by examining either DNA or protein adduct levels (33, 64, 65). Protein adduct levels are elevated in a variety of diseases associated with oxidative injury (e.g., Parkinson’s disease, Alzheimer’s disease, and atherosclerosis) (66-68). A recent quantitative study examining the conjugation of HNE to vitamin C suggests that this conjugate exists at concentrations between 1 and 5 µM in the plasma of healthy human subjects (35). Because numerous reactive intermediates are produced during oxidative stress as outlined above, it is likely that they collectively accumulate to levels capable of altering biological processes and function in concert to bring about adverse cellular effects, including alteration of cell stress signaling and eventually cell death.

3. Cell Death Mechanisms Activated by Endogenous Reactive Intermediates Reactive intermediates produced during the process of oxidative stress represent a broad panel of endogenously produced cytotoxic agents. Cell death linked to oxidative damage is observed in vivo following ischemic injury and in agingassociated neurodegeneration (3, 4, 69, 70). The spectrum of cell death observed under oxidative injury circumstances ranges from necrotic to apoptotic death.


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3.1. Necrosis Necrosis is a disordered mode of cell death, which is often described in terms of morphological changes. A prominent characteristic that necrotic cells display is membrane rupture, typically after exposure to pronounced toxin concentrations or physical insult (1, 71). Upon exposure, most cellular barrierss including the plasma, nuclear, mitochondrial, and endoplasmic reticulum (ER) membranessswell as a result of altered ion homeostasis and eventually rupture (72, 73). Along with the loss of membrane integrity, there is a rapid decrease in the capacity to synthesize ATP, due to the demise of mitochondrial electron transport and membrane potential (71, 73). Likewise, leakage of lysosomal proteases outside of the cell corpse occurs, and the ensuing inflammatory response in tissues serves to promote healing and clearance of damaged cells (73, 74). The process of necrosis is tightly regulated in certain instances. Proteins such as poly(ADP-ribose) polymerase-1 (PARP-1) can execute immediate cell death following activation by necrotic stimuli, including high doses of hydrogen peroxide and peroxynitrite (75-77). In murine models for ischemic brain injury and diabetes, PARP-1 is a critical determinant of extent of injury (76, 78-81). The involvement of PARP-1 in such responses is presumed to occur through its ability to induce necrotic death. Because many reactive intermediates are produced following ischemic injury (e.g., stroke, myocardial infarction) and diabetic degeneration, DNA damage stimulated by these molecules is often offered as an explanation for extensive PARP-1 activation and subsequent necrotic cell death (70, 77). The mechanism through which PARP-1 mediates such effects is still under intense scrutiny; it causes rapid depletion of NAD and ATP and may involve the release and recruitment of apoptosis-inducing factor from mitochondria to the nucleus for degradation of genomic DNA (75, 82, 83). Because there are few signature biochemical features for this mode of necrotic death, it is sometimes referred to as caspase-independent apoptosis, although the overall phenotype is similar to traditional necrosis. Although recent reports suggest regulatory mechanisms for necrosis, this is not necessarily of broad application to all forms of necrotic death. A clearly defined and unified molecular model for necrotic death, therefore, remains elusive.

3.2. Apoptosis The term “apoptosis” is derived from the Greek word for “leaves falling from a tree”, a distinctly ordered event taking place every autumn (72). It occurs as a well-defined process in all higher eukaryotes, as is indicated by the fact that it was jointly discovered in mammalian cells and organisms used for genetic manipulation. In nonmammalian model systems, the phrase “programmed cell death” was coined to account for apoptotic death occurring at specific stages during organism development (84, 85). The majority of the biochemical discoveries in apoptosis has been made in the past 15 years (86). Perhaps the most essential finding during this period has been that most forms of apoptotic death hinge upon the activation of a family of cysteine proteases called caspases (87, 88).

3.2.1. Caspases Caspase-1 was originally identified as the cysteine protease responsible for converting prointerleukin-1β (a cytokine) to its active form; therefore, it was named interleukin-1β-converting enzyme (ICE) (89, 90). A significant breakthrough in cell death research was the discovery that ICE and the Caenorhabditis

Figure 4. Three-dimensional structure of caspase-3 bound to a covalent inhibitor. Activated caspase forms a heterotetramer, resulting from the cleavage of two procaspase monomers. The large subunits (shown in blue) contain the catalytic cysteine residue (shown in green covalently linked to the DVAD-CHO inhibitor shown in red). The small subunits provide the majority of the interface between the tetramer and, together with the large subunits, form an inner core β-sheet. This depiction of caspase-3 (pdb 1CP3; ref 319) was generated using UCSF Chimera software.

elegans CED-3 protein (a protein critical for programmed cell death) share considerable sequence homology, suggesting that ICE-related proteins play a critical role in apoptosis in addition to cytokine processing (91, 92). Through homology comparisons, several ICE-related cysteine proteases have been identified as key factors involved in executing the apoptotic program (93). The term caspase, standing for a “cysteinyl-directed aspartatespecific protease”, has been adopted as the universal name for individual members of the ICE family of enzymes (94). Caspases are tightly regulated in cellular environments, depending on specific signals to stimulate their activity. The human caspase gene family plays diverse roles in regulation of apoptosis and inflammation (87, 95). Caspases normally exist as zymogenssinactive proenzymes requiring dimerization and/or cleavage for proper activation (89, 90). The cleaved forms of caspases function as heterotetramers and demonstrate distinct cleavage specificity for peptide substrates (Figure 4). Such target selectivity is dictated by consensus amino acid sequences in substrates (96). In addition, defined patterns of caspase activation depend on individual apoptotic agonists, allowing for integration of various extracellular and intracellular stress signals. Specifically, apoptotic caspases are divided into initiator or effector enzymes, based on whether they possess caspase recruitment or death effector domains (which are present in initiator caspases) and upon the order of their activation (87, 95). During apoptosis, initiator caspases are stimulated intrinsically by release of mitochondrial components or extrinsically by extracellular signaling proteins (e.g., TNFR, FasL), leading to cleavage and activation of effector caspases.

3.2.2. Mechanisms of Caspase Activation Extensive exposure to most reactive intermediates often results in the internal, or intrinsic, activation of caspase-mediated signaling. This pathway is triggered by release of several proapoptotic proteins from mitochondria into the cytosol (Figure 5) (97). These factors include cytochrome c, second mitochondrial activator of caspases/direct IAP binding protein with low pI (Smac/DIABLO), and Omi/HtrA2. These proteins play a

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Figure 5. Apoptotic pathway activation. Intracellular activation of caspases can be triggered by many endogenous reactive intermediates through release of cytochrome c from mitochondria. Some caspase activation may also be the result of ER stress signaling and/or alteration of gene expression of factors involved in extrinsic pathway activation. Once caspases are activated, a variety of cellular targets are cleaved, resulting in the biochemical and cytological changes described at the bottom.

prominent role in promoting caspase activation, and their release from mitochondria is regulated by a common, although unresolved, mechanism. A family of related proteins is involved in regulating the permeability of mitochondria during apoptosis, allowing for release of the above-mentioned proapoptotic components. The protein encoded by the Bcl-2 (B cell lymphoma-2) oncogene [originally discovered as part of chromosomal translocation t14;18 in lymphomas (98-100)] is a negative regulator of apoptotic progression (101, 102). Bcl-2 localizes directly to mitochondrial membranes in intact cells (101, 103) and prevents apoptotic death upon overexpression. In biochemical experiments using Xenopus laeVis egg extracts, the addition of mitochondrial fractions to cytosolic lysates of apoptotic cells enhances chromatin condensation and DNA fragmentation in intact nuclei, both of which are prevented by the addition of recombinant Bcl-2 (104). From these findings, it was deduced that proteins residing in mitochondria play a pivotal role in activation of the apoptotic program. Although there is a great deal of uncertainty and controversy concerning how mitochondria release cytochrome c, Smac/ DIABLO, and Omi/HtrA2, Bcl-2 and closely related antiapoptotic proteins (e.g., Bcl-XL) prevent this process (105-108), whereas distant proapoptotic relatives of Bcl-2 (e.g., Bax, Bak, and Bim) promote mitochondrial permeabilization (109-111). Several hypothetical models involving antiapoptotic and proapoptotic Bcl-2 family members may account for mitochondrial permeabilization. Specifically, the processes of mitochondrial

fission or the formation of a permeability transition pore involving either proapoptotic Bcl-2 family proteins, voltagedependent anion channels, and/or the adenine nucleotide transporter are potential modes of proapoptotic protein release into the cytosol (110, 112). Currently, it is unclear which model is the most accurate, as there is support for each. Once present in the cytosol, cytochrome c binds to apoptotic protease activating factor-1 (Apaf-1) (113, 114). Cytosolic extracts supplemented with ATP or dATP and cytochrome c cause activation of caspase-3, nuclear condensation, and DNA fragmentation in intact nuclei (113, 115). During this process, Apaf-1 heptamerizes and interacts with cytochrome c and procaspase-9 (116). The assembly of this complex, called the apoptosome, is a major converging point through which the intrinsic apoptotic pathway is induced. Upon binding of ATP or dATP by the apoptosome, two molecules of procaspase-9, an initiator caspase, are brought into close proximity of one another and dimerize, undergoing autoproteolysis enabling them to cleave downstream effector caspases (113, 114, 117-119). Subsequently, effector caspases (e.g., caspase-3, -6, and -7) execute the final proteolytic stage of apoptosis (120). Although cytochrome c is a crucial component of the electron transport chain, its release from mitochondria during apoptosis reveals a role of equal importance in the promotion of apoptotic cell death (121). Other factors released from mitochondria promote caspase activation by preventing caspase inhibition. Specifically, Smac/ DIABLO and Omi/HtrA2 antagonize the function of various


inhibitors of apoptosis proteins (IAPs), a family of proteins which directly bind to and inhibit initiator and effector caspases (118, 122). Smac/DIABLO counteracts IAP-mediated caspase inhibition by binding to the baculoviral-inverted repeat-3 domain in IAPs, a sequence that shares significant structural homology to a domain in caspase-9 (118, 123-126). Omi/HtrA2 directly cleaves XIAP to render it incapable of inhibiting caspase activity (127). Using independent means of IAP inactivation, both mitochondrial proteins enhance activation of the intrinsic caspase pathway. Caspases-2, -4, and -12 are also involved in the intrinsic apoptotic pathway, although their functions are less clearly defined. Caspase-2 is required for apoptotic signaling in response to genotoxic stress (128, 129). Its activation is stimulated by way of a large assembly complex, recently identified as the PIDDosome (130, 131). The PIDDosome, a macromolecular scaffold, consists of caspase-2, a p53-regulated gene product PIDD, and an apoptotic adaptor protein, RAIDD (131-134). Caspase-2 functions as an initiator caspase following irreparable DNA damage, acting directly upstream of mitochondria to stimulate the release of cytochrome c and other components through an undescribed mechanism (128, 129, 135). Murine caspase-12 and human caspase-4 may function as ER stressresponsive caspases (136, 137), although the human CASP-12 gene does not encode an active protease (138) and these results have been questioned by others (139). How caspases are activated in response to disruption of ER homeostasis by reactive intermediates and what role Bcl-2 family members, which localize to the ER as well as mitochondria, have in regulating this process are both currently areas of active exploration. Membrane receptors can also transduce extracellular apoptotic signals (e.g., TNFR, FasL) via stimulation of the extrinsic caspase pathway (140-142) (Figure 5). Specifically, TNFR and FasL induce caspase activation through adaptor molecules that form a caspase-8 or -10 complex at the membrane (143-147). Upon activation, caspases-8 and -10 cleave effector caspases directly (148) but also promote increased activation of the intrinsic pathway. Cross-talk with the intrinsic pathway is mediated by cleavage of the BH3 only protein, Bid, to its truncated form, tBid (149-151). tBid migrates to mitochondria, allowing for permeabilization and proapoptotic protein release (150, 151). It is likely that some signal amplification during apoptosis induced by reactive intermediates is the consequence of extrinsic pathway signaling.

3.2.3. Caspase Targets Upon activation, effector caspases (e.g., caspases-3, -6, and -7) digest numerous cellular proteins and function in most subcellular locations. Specifically, proteolysis of targets in the cytosol, mitochondria, and nuclei gives rise to many of the characteristic morphological and biochemical features of apoptosis. Cleavage of most targets is not necessary but, in some cases, may be sufficient for completion of apoptotic signaling. Although not a comprehensive list (for one, see ref 88), the caspase targets discussed represent those which are of historical importance and/or those which provide a biochemical explanation for particular apoptotic events (Figure 5). 3.2.3.1. Cytoskeletal and Cytosolic Caspase Substrates. Many of the cytosolic targets of effector caspases are cytoskeletal components, and presumably, their cleavage results in membrane blebbing, loss of cell adhesion, and apoptotic body formation. Specifically, caspases cleave the cytoskeletal components R-fodrin, Gas2, β-catenin, ROCK1, PAK2, and gelsolin (152-163). PAK2 and ROCK1 function as kinases that regulate

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actin filament stabilization; cleavage by caspase-3 significantly enhances their kinase activity (158-161, 163). Ectopic expression of the cleaved forms of Gas2, gelsolin, PAK2, and ROCK1 causes cytoskeletal rearrangement and membrane blebbing similar to that seen in apoptotic cells (155, 158-160, 162, 163). Proteolysis of these caspase substrates is not necessary for completion of apoptotic signaling but may instead facilitate membrane blebbing, formation of apoptotic bodies, and engulfment by surrounding cells. In addition to the kinases PAK2 and ROCK1, several protein kinase C isoforms are cleaved and activated by caspases, leading to some of the phenotypic indicators of apoptosis (164-168). The kinase substrates responsible for these effects are undefined, although their phosphorylation may play a broad role in regulating phosphatidylserine exposure, cytoskeletal rearrangement, and nuclear apoptotic events (166, 167, 169). 3.2.3.2. Mitochondrial Caspase Substrates. Since the discovery of cytochrome c release and loss of mitochondrial membrane potential during the initiation phase of apoptosis, mitochondrial function has been a proposed target of caspase activity (170, 171). Indeed, treatment of permeabilized mitochondria with caspase-3 causes a rapid loss of electron transport, oxygen consumption, and transmembrane potential by inhibiting the function of complexes I and II and leads to an increase in ROS production by mitochondria (172). In a proteomic search for caspase-3 substrates in mitochondria lysates, the p75 subunit of complex IsNADH dehydrogenase Fe-S protein-1swas identified and verified to contain an internal caspase-3 consensus cleavage sequence (173). Cleavage of this subunit causes a decrease in both electron transport and oxygen consumption in mitochondria, reduces overall ATP levels in cells, and allows for increased externalization of phosphatidylserine in apoptotic cells (173). 3.2.3.3. Nuclear Caspase Substrates. In addition to cytoplasmic and mitochondrial substrates, numerous nuclear proteins are cleaved by caspases. These proteins can be arbitrarily grouped into one of several categories, including nuclear structural proteins and proteins involved in DNA metabolism and repair. Nuclear structural components cleaved by caspases include nuclear lamins A/C and B1. Lamins A and C are thought to be the exclusive targets of caspase-6 (174, 175), whereas the caspase cleaving lamin B1 is not known (176). Cleavage of lamins promotes loss of nuclear structure, a traditional apoptotic indicator, and may influence nuclear condensation and chromatin degradation in apoptotic cells in a coordinated effort with the factors described below (177, 178). Additionally, several proteins involved in DNA metabolism are cleaved by caspases in the terminal stages of the apoptotic process. Cleavage of two of thesesDNA fragmentation factors 45 and 35 (DFF45/DFF35)sinduces apoptotic nucleosomal fragmentation (179, 180). DFF45 and a truncated splice variant DFF35 function as inhibitors of the predominant apoptotic nuclease, DFF40 (181-183). During apoptosis, DFF45 and DFF35 are cleaved by caspases, leading to the activation of DFF40 and the fragmentation of genomic DNA into 50-300 kilobase pair and nucleosomal (i.e., 180 base pair) products (180, 182). At the same time of DFF40 activation, several enzymes that respond to DNA strand breaks are inactivated by caspases-3 and -7. These include PARP-1, ataxia-telangectasia mutated protein, and the catalytic subunit of DNA-dependent protein kinase (93, 184-189). As apoptotic cells undergoing DNA fragmentation show signs of activation of these enzymes (190192), their cleavage may facilitate the degradation of genomic DNA and prevent any counterproductive DNA repair. Addition-

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Figure 6. Antioxidant response signaling activation. Nrf2, the master regulator of the response, is retained in the cytosol by its inhibitory protein, Keap1, which also interacts with the ubiquitin ligase, Cullin 3. In the presence of reactive intermediates, Keap1 is modified, promoting release of Nrf2 and allowing Nrf2 to migrate into the nucleus to stimulate expression of genes, which protect against oxidative damage.

ally, activation of the uncleaved enzymes may result in direct inhibition of DFF40, thereby blocking genomic DNA degradation, as has been suggested for PARP-1 (191).

4. Cell Signaling Pathways Influenced by Reactive Intermediates Which Alter Cell Death The signaling events that occur upstream of apoptotic commitment make understanding the apoptotic process a complicated matter. A panoply of factors influence death by modulating the effects of apoptotic signaling proteins directly and by affecting expression of proapoptotic or cytoprotective genes. Several specific examples of signaling cascades that are affected by reactive intermediates, many of which have been identified through gene expression profiling experiments for H2O2 and HNE (193-197), will be discussed, with particular emphasis being placed on their ability to alter cellular viability.

4.1. Antioxidant Response Signaling Many reactive intermediates, including H2O2 and HNE, activate the antioxidant, or phase II, response. This signaling cascade culminates in the nuclear translocation of and transactivation by the transcription factor, Nrf2 (198, 199) (Figure 6). Nrf2 activity is repressed by an inhibitory binding protein, Keap1, via several interrelated mechanisms (200). Keap1 retains Nrf2 in the cytosol, closely associated with the actin cytoskeleton (201, 202), and promotes proteasomal degradation of Nrf2 through Cullin3-dependent polyubiquitination (203-206). Following exposure to oxidants and alkylating agents, Keap1 is directly modified on several cysteine residues, and this modification can promote release of Nrf2 under some experimental conditions in vitro (207-209). Intracellular modification of Keap1 similarly allows for increased stabilization and nuclear localization of Nrf2, potentially through direct modification of Keap1 coupled with Keap1 ubiquitination and degradation (210-212). Once in the nucleus, Nrf2 works in conjuction with small Maf proteins to regulate a battery of genes that protect against oxidative injury (198, 213). Nrf2 activity is also enhanced through phosphorylation by several kinases, including protein kinase C isoforms and the ER-stress responsive kinase PERK (214-216).

Using traditional approaches as well as gene expression profiling, a large number of Nrf2 target genes have been identified (217-219), and many of them play defined roles in facilitating protection against and recovery following oxidative injury. For example, subunits of glutamate-cysteine ligase (GCL) and of the Xc- cystine transporter are regulated by Nrf2 to accelerate production of GSH (220-223). Consistent with this notion, induction of these genes raises intracellular GSH levels to protect against oxidative injury (209, 223). Other Nrf2 target genes include those encoding proteins involved in protein ubiquitination and proteosomal degradation and enzymes responsible for inactivating oxidizing and alkylating agents [e.g., NADH-dependent quinone oxidoreductase-1 (NQO1), GSTs, AKRs, and heme oxygenase-1 (HO1)] (198, 217-219). If Nrf2 target genes are induced prior to treatment with toxic doses of hydrogen peroxide, the extent of cell death is dramatically reduced, implying that Nrf2 activation promotes a cytoprotective response (218, 224). Therefore, stimulation of this pathway by subcytotoxic doses of reactive intermediates may serve to prevent their accumulation to toxic levels.

4.2. Heat Shock Response Signaling The heat shock response is stimulated following exposure to virtually all reactive intermediates, in addition to many other noxious stimuli (225). Specifically, heat shock gene expression is stimulated by endogenous oxidants and lipid peroxidation products alike. Activation of heat shock factor-1 (HSF1) during this process subsequently induces the expression of a variety of heat shock proteins (Hsps) (Figure 7). HSF1, like Nrf2, resides predominantly in the cytosol under basal conditions, where it is bound and inhibited by various Hsps, including Hsp90, Hsp70, and Hsp40 (226-228). A potential hypothesis is that, following extensive protein damage by reactive intermediates, Hsps are recruited to sites of protein damage and therefore release HSF1, thereby promoting its activation. This sequence of events allows HSF1 to migrate into the nucleus, trimerize, undergo hyperphosphorylation, and stimulate transcription of Hsp genes (225). HSF1 regulation is also redox sensitive, with its activation potentially requiring formation of a disulfide bond (229). When active, trimeric HSF1 binds to inverted repeats of the sequence 5′-nGAAnnTTCn in the promoters of numerous Hsp genes, giving rise to downstream effects that influence both protein folding homeostasis and cell death (225). Of the molecular chaperones regulated by HSF1, Hsp70 is currently the best understood regarding its regulation of signaling and cell death pathways. In addition to its function of promoting refolding or clearance of misfolded or aggregated proteins (230), Hsp70 prevents apoptosis through disruption of apoptosome formation, potentially through directly binding Apaf1 (231, 232). An additional mode through which Hsp70 potentially inhibits cell death is by blocking stress-responsive mitogen-activated protein kinase (MAPK) signaling (see below for explanation of these kinases) (233, 234). Therefore, specific Hsp70 isoforms induced in stressed cells following HSF1 activation may function as negative regulators of apoptotic signaling in multiple ways.

4.3. Nutrient Deprivation and ER Stress Response Signaling Several transcription factors coordinately regulate gene expression changes and cell fate decisions following ER stress and amino acid deprivation (235, 236) (Figure 8). The activation of these factors potentially results from direct damage of proteins


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Figure 7. Heat shock response activation. HSF1 is normally inhibited by the molecular chaperones, Hsp70 and Hsp90. Following accumulation of damaged or misfolded proteins, HSF1 trimerizes, undergoes posttranslational modification, and stimulates expression of many Hsp genes.

Figure 8. ER stress/nutrient deprivation response signaling. Reactive intermediates may disrupt ER homeostasis through altering protein folding homeostasis or depleting amino acids, leading to the activation of the transcription factors ATF4, XBP1, and ATF6. The target genes of each transcription factor play diverse roles in restoring ER homeostasis, promoting amino acid import and biosynthesis, and influencing cell death decisions.

being translated and folded in the ER by reactive intermediates, as this response is regulated by the ER-resident Hsp70 homologue, glucose regulated protein 78/immunoglobulin binding protein (Grp78/BiP) (236). Additionally, as reactive intermediates deplete certain amino acids (e.g., Cys) during their

detoxification, an unfavorable environment for protein translation and folding potentially results. The mechanism through which Grp78/BiP is activated during perturbation of ER homeostasis is therefore complicated. In general, ER stress results in the initial activation of at least three ER stress-

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responsive transcription factors [i.e., activating transcription factor 4 (ATF4), X-box binding protein 1 (XBP1), and ATF6] through independent mechanisms described below (236). The transcription factors ATF6 and XBP1 function in response to a variety of exogenous ER stressors (e.g., tunicamycin, thapsigargin) but may be influenced as well by reactive intermediates produced endogenously. ATF6 is synthesized as a transmembrane proprotein and is normally bound to BiP/ GRP78 (237). Upon disruption of ER homeostasis, ATF6 is transported to the Golgi and cleaved to form a mature, functional transcription factor by Golgi proteases S1P and S2P (237). Once cleaved, ATF6 binds to ER stress response elements (ERSE) in target genes to induce several genes that promote proper protein folding in ER, including a number of ER-resident molecular chaperones BiP/Grp78 and Grp94 and the transcription factor XBP1 (238-241). XBP1 is activated by ER stresses following the release of the splicing factor IRE1 from inhibition by BiP/Grp78 (242). IRE1 promotes proper processing of the XBP1 transcript, allowing its translation as a transcriptional activator to promote recovery of ER homeostasis through binding the unfolded protein response element (UPRE) (238). Like ATF6, XBP1 strongly promotes expression of ER-resident molecular chaperones. Another prominent feature of ER stress is that protein translation is temporarily halted with a few notable exceptions. This occurs through direct phosphorylation of the translation initiation factor eIF2R via one or more of several different kinases (e.g., PERK, GCN2) (243, 244). Although most routine protein synthesis is disrupted, the ATF4 transcript is selectively translated (245, 246). ATF4 promotes recovery from amino acid depletion by increasing the levels of various amino acid transporter subunits (e.g., xCT, SLC3A2) and genes involved in amino acid metabolism and biosynthesis (e.g., asparagine synthetase) (247-249). Additionally, ATF4 controls transcription of several ER stress-responsive genes that alter apoptotic signaling (e.g., CHOP/Gadd153, TRB3) (248, 250). In response to reactive intermediates, the ATF4 arm of the ER stress pathway is perhaps the most strongly induced; yet, many features of this stress response mechanism remain undefined.

4.4. Stress Responsive MAP Kinase Signaling The stress-responsive MAPKs c-Jun N-terminal kinase (JNK) and p38 enhance apoptotic signaling (251). Under homeostatic conditions, isoforms of JNK and p38 are activated by various stimuli (e.g., phorbol esters, TNFR) and phosphorylate leucine zipper transcription factors (e.g., AP-1), thereby stimulating transcription of many different immediate early genes (252). Likewise, exposure to various reactive intermediates results in prolonged activation of JNK and p38, leading to augmentation of apoptotic signaling (Figure 9). It is currently unclear how both JNK and p38 bring about such effects on cell death. It is postulated that JNK isoforms phosphorylate and potentially inactivate Bcl-2 and Bcl-xL (253-256). Additionally, JNK may phosphorylate and activate proapoptotic Bcl-2 family members (e.g., Bim, Bad) to stimulate mitochondrial permeabilization (257, 258). Although there is confusion regarding the exact mechanism of JNK apoptotic regulation, there are numerous correlative reports linking JNK activation to induction of apoptosis by reactive intermediates (259-263). It is conceivable that these effects are mediated through phosphorylation and activation of its transcription factor targets, through direct effects on mitochondrial permeability, and/or through other mechanisms. In recent years, at least two different hypotheses have emerged to account for activation of JNK and p38 by reactive intermedi-


Figure 9. Stress-responsive MAPK signaling. Stress-responsive MAPK signaling potentially is stimulated in multiple ways, including inactivation of MKPs by reactive intermediates or direct stimulation of MAPKs. Transcriptional activation by various leucine zipper (LZ) transcription factors occurs, as well as phosphorylation of mitochondrial targets. Both of these processes may lead to mitochondrial permeabilization and apoptotic initiation.

ates. The first is that these kinases are directly modified byproducts of oxidative injury, although only one instance of this has been reported. HNE purportedly reacts directly with JNK to stimulate its activity under some experimental conditions (264). However, another likely possibility is that most electrophilic and oxidizing products of oxidative injury directly inactivate MAPK phosphatases (MKPs) (265). These dual specificity enzymes inactivate JNK and p38 by catalyzing their dephosphorylation on both Thr and Tyr residues. MKPs, like other tyrosine phosphatases, possess a strongly acidic catalytic Cys residue (pKa ∼ 5.5), which is sensitive to direct oxidation or other modification (266, 267). Inactivation of MKPs would therefore allow for prolonged activation of stress-responsive kinases and would potentially enhance their apoptotic effects.

4.5. NF-KB Signaling The transcription factor NF-κB is activated in response to various inflammatory cytokines, phorbol ester, and calcium flux (268, 269). Following treatment with these stimuli, a kinase cascade leads to the phosphorylation of the inhibitor of NF-κB, IκBR, by IκB kinase (IKK) (268, 269) (Figure 10). Subsequently, IκBR is degraded, and NF-κB is released into the nucleus where it stimulates the transcription of immediate early and antiapoptotic genes. Several antiapoptotic genes are positively regulated by NF-κB, including cIAP and antiapoptotic Bcl-2 family members, Bfl-1 and Bcl-XL (270-272). Additionally, NF-κB induces several genes involved in preventing activation of the extrinsic apoptotic pathway (270). NF-κB may also prevent apoptosis through antagonism of proapoptotic, stress-responsive MAPK signaling (273-275). In response to reactive intermediates, stimulus-induced NFκB signaling can be disrupted. NF-κB activation by various inflammatory stimuli is strongly inhibited in the presence of H2O2, HNE, acrolein, and other R,β-unsaturated carbonyls, and this is likely to occur through direct modification of a key regulatory Cys residue on IKK, which leads to loss of kinase activity (276-279). While other reports confirm the overall inhibition of NF-κB signaling by these molecules, the same effects on IKK activity have not been observed (280, 281). The reason for the discrepancy likely lies in the nature of the experimental approach for IKK activity assays. In the absence of reducing agents, IKK is modified and inactive; however, in the presence of dithiothreitol, these modifications are reversed,


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Figure 10. NF-κB signaling. IKK is activated by external stimuli such as inflammatory cytokines, leading to the phosphorylation, ubiquitination, and degradation of IκBR. The NF-κB complex, p65/p50, migrates into the nucleus and stimulates expression of anti-apoptotic genes. Reactive intermediates directly inhibit this pathway by modifying a critical Cys residue on IKK.

so little effect on IKK activity is often observed under these conditions (277). Nonetheless, many reactive intermediates can negatively influence this important cellular survival pathway.

4.6. DNA Damage Response Signaling Few stress-responsive transcription factors have received as much attention as the p53 protein. The p53 gene encodes a tumor suppressor protein, and its activity is disrupted in many cancers (282). As a critical controller of cellular responses to stress, p53 activation leads most often to cell cycle arrest (to repair damaged DNA) and/or apoptosis (282) (Figure 11). p53 is normally inhibited by Mdm2-stimulated ubiquitination and proteasome-mediated degradation. Following excessive DNA damage, p53 is released from Mdm2, is stabilized by phosphorylation, acetylation, and other posttranslational modifications, and undergoes tetramerization to bind its DNA sequence (282). p53 mediates many of its biological effects by increasing transcription of genes which negatively regulate cell progression (e.g., cyclin-dependent kinase inhibitor, p21/WAF1/Cip1, and Cdc25 inhibitor, and 14-3-3σ) or of genes that promote apoptotic signaling (e.g., Bax, Noxa, Fas, and Apaf-1) (282, 283). p53 is recognized as a key regulator of DNA damage-induced gene transcription leading to both cell cycle arrest and/or apoptosis. In addition to its effects on transcriptional regulation, p53 localizes to the cytosol and/or mitochondria to induce apoptosis through the intrinsic pathway. Several lines of evidence support such a role. p53 can induce apoptosis in the absence of de novo gene expression, suggesting that transcriptional activation represents only a partial explanation for its apoptotic effects (284). Following treatment with apoptotic stimuli, p53 accumulates in the cytosol (and potentially in mitochondria membranes) to promote cytochrome c release through cooperation with Bax and other proapoptotic Bcl-2 family members (285-289). Although p53 promotes apoptosis via its function as a transcriptional activator, it functions through transactivationindependent mechanisms as well. Reactive intermediates have opposing effects on p53 signaling, because they act both as DNA damaging and protein modification agents. Most reactive intermediates cause ac-

Figure 11. DNA damage signaling by p53. DNA damage by reactive intermediates may result in p53 activation through a process that involves its release from Mdm2 and subsequent stabilizing posttranslational modifications. However, certain reactive intermediates may also disrupt p53 activation by modifying and inhibiting TrxR and/or Trx. When active, p53 promotes expression of genes involved in DNA repair, cell cycle arrest, and apoptotic progression. Additionally, p53 can relocalize in the cell to exert direct effects on mitochondrial permeabilization, promoting cytochrome c release and caspase activation.

cumulation of p53, potentially through their ability to damage DNA directly (290). However, the cytotoxic response that they initiate is not necessarily p53-dependent (291, 292). On the basis of these findings, it is assumed that p53 is not entirely responsible for stimulating reactive intermediate-induced apoptosis. In fact, stimulus-induced p53 activation is strongly inhibited by R,β-unsaturated carbonyls (293, 294), and this is

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due predominantly to direct modification of enzymes in the thioredoxin (Trx)/thioredoxin reductase (TrxR) system (294, 295). Therefore, it is possible that the excessive p53 accumulation and activation needed for apoptotic induction is limited by reactive intermediates, accounting for its nonessential role in promoting caspase activation under certain conditions.

5. Complexity in Oxidative Stress Signaling: Lessons Learned from HNE HNE is one of the most reactive and widely studied cytotoxic products of lipid peroxidation, and it is formed in vivo during periods of oxidative damage. The cellular effects of HNE as they relate to cytotoxicity have been examined using multiple approaches and reveal a complicated series of signaling events that converge to influence cell cycle control and cell death.

5.1. Cellular Responses Induced by HNE The mechanisms through which HNE induces cell death vary in a cell type and dose-dependent manner. Over a narrow concentration range, HNE causes apoptosis, whereas when this threshold dose is exceeded, necrosis results (296, 297). In cell types where apoptosis is prevalent, HNE typically activates caspases via the intrinsic pathway. Ectopic expression of Bcl-2 prevents apoptosis in response to HNE by blocking proapoptotic protein release from mitochondria (292, 297, 298). The reactive properties of R,β-unsaturated aldehydes play an essential role in stimulating cell death cascades, and a correlation between the electrophilic and the cytotoxic strength of these molecules has been observed in many, but not all, instances (59, 298303) (Ji, C., West, J. D., and Marnett, L. J. Unpublished observations). The apoptotic response stimulated by HNE requires de novo gene expression (298), which prompted us to examine the global gene expression changes and the stress signaling effects of HNE (197). From such studies, we observed that subcytotoxic and cytotoxic concentrations of HNE induce multiple changes in gene expressionsboth increases and decreasessin human RKO colorectal carcinoma cells, with the highest level of changes being observed at high HNE doses at times before the cells undergo apoptosis. Several key signaling pathways linked to apoptosis, including heat shock response activation, NF-κB inactivation, JNK activation, and p53 target gene expression, are significantly influenced by HNE, as has been indicated through traditional analysis of individual cell signaling pathways (56, 264, 277, 281, 292, 304). Using microarray technology, we found that the most significant alterations in gene expression occurred in gene families regulated by the antioxidant, heat shock, and amino acid deprivation responses (197). Collectively, these results imply that HNE simultaneously stimulates multiple stress signaling responses. Many of these represent potential mechanisms through which HNE could alter cellular viability. Numerous cellular detoxification pathways exist to limit the accumulation of HNE, and these enzymes play an important role in reducing or completely preventing HNE-mediated cell death. Ectopic expression of GSTA4-4, aldehyde dehydrogenase 3, and 15-oxoprostaglandin-13-reductase markedly reduces HNE-mediated protein damage and cytotoxicity (59, 61, 260). Increasing intracellular levels of GSH with N-acetylcysteine decreases the cytotoxic effects of HNE (305), whereas preventing GSH biosynthesis by treatment with buthionine sulfoximine enhances HNE-mediated cell death (306, 307). Together, these reports confirm that multiple pathways exist to protect against the cytotoxic effects of HNE and related molecules produced during oxidative stress.

Many of the metabolic enzymes that directly or indirectly provide protection against HNE-mediated death are induced at the level of transcription by activation of the antioxidant response. Nrf2 activation occurs following exposure to even subcytotoxic levels of HNE (197), presumably through the direct modification and inactivation of Keap1 (209). Subcytotoxic doses of HNE promote an adaptive response that preconditions cells for tolerance against hydrogen peroxide-mediated death (224). Therefore, activation of this pathway by low levels of HNE or other inducers in vivo may have beneficial effects in protecting cells against numerous reactive molecules produced during oxidative injury.

5.2. Macromolecular Targets of HNE The toxicity and mutagenicity of HNE are derived from its chemical reactivity. Thus, an important goal is identification of the macromolecular targets of HNE, particularly those responsible for its biological activities. DNA damage is presumably responsible for the mutagenicity of HNE and also may contribute to its cytotoxic and cytostatic effects, particularly through activation of the p53 DNA damage response (292) (Ji, C., West, J. D., and Marnett, L. J. Unpublished observations). However, the activation of p53 by HNE may be short-lived and limited in scope. Although the classical pathway of p53 stabilization and accumulation is dependent on DNA damage, there are other pathways for modulation of p53 that result from protein modification. Electrophilic aldehydes and ketones, including HNE, indirectly generate a transcriptionally inactive conformation of p53. This inactive form of p53 is likely to result from direct modification and inactivation of TrxR, a selenocysteine-containing enzyme that controls the conformation of a number of redox-sensitive proteins (294). Therefore, TrxR inhibition by HNE may account for the lack of p53 dependence in the apoptotic response that it stimulates in certain cell types (292). Protein modification by lipid-derived aldehydes, including HNE, potentially plays an important role in the toxicity associated with many pathological conditions, including alcoholic liver disease (308). In rats exposed to a high fat diet supplemented with alcohol, protein adducts of HNE are detected throughout the liver (308). Two-dimensional profiling of HNEadducted proteins in cytosolic and membrane fractions has revealed several modified proteins, some of which are likely to play a role in the cytotoxic response. The modified proteins include the chaperones Hsp90 and Hsp72 and protein disulfide isomerase (309-311). Modification of all three proteins occurs principally on cysteine residues, although it is likely that other, unidentified modification sites exist. Cysteine modification correlates with the loss of the ability of the Hsps to bind heatinactivated proteins and the loss of protein disulfide isomerase catalytic activity (309-311). The combined action of HNE on these three proteins (and possibly other related proteins) could significantly alter productive protein folding and trafficking and lead to reduced viability. In addition, various Hsps, including Hsp90 and Hsp70, bind the transcription factor, HSF-1, keeping it in a transcriptionally inactive state (226-228). A hypothetical explanation for HSF-1 activation by HNE could include inactivation of Hsp90, which would allow HSF-1 to undergo trimerization and nuclear localization. Also, Hsp90 and Hsp70 bind to Apaf-1 and inhibit apoptosis by preventing assembly of the apoptosome (231, 232, 312). Modification of Hsp90 and Hsp70 may sensitize cells to HNE-induced cytotoxicity by eliminating their interactions with Apaf-1.


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Figure 12. Collective alteration of cell signaling networks by endogenous reactive intermediates. Most reactive intermediates arise, presumably, from aberrant mitochondrial respiration. Following their production, they have multiple intracellular targets, including small nucleophiles (e.g., GSH, amino acids), proteins, and DNA. Modification of these targets brings about a number of cell signaling responses and may ultimately lead to cell death.

6. The Future The pleiotropic effects of reactive intermediatessoxidants, nitrosating and nitrating agents, and aldehydessmost likely result from modification of multiple intracellular targets (Figure 12) and may vary depending on the individual damaging agents and cell type(s) being utilized. Therefore, it may be unrealistic to search for a single modified protein that is responsible for biological effects that are inherently complex in mechanism and control (e.g., apoptosis). The global pattern of protein and DNA modification may determine the ultimate response of the cell. Proteomic approaches permit global analysis of protein modification, and an abundant amount of such information will be acquired in the relatively near future. Although the ultimate cellular response may result from multiple macromolecular modifications, it will be of great importance to evaluate the functional effects of modifying individual proteins. Complementary approaches to altering the abundance and function of individual proteins based on ectopic expression or siRNA knockdown are now straightforward. Additionally, the generation of target proteins bearing site-directed mutants at residues susceptible to modification will allow for probing the importance of such modifications in cell function directly. Identification of other toxic lipid oxidation products will be an additional area of emphasis in coming years. Traditionally, work on lipid-derived aldehydic products has centered on diffusible products of lipid peroxidation. However, there are many products of lipid peroxidation in which the reactive functional group remains bound in the phospholipid residue (313). The ability of such molecules to react with targets and exert cytotoxic effects is only beginning to be investigated. For example, different oxidation products of phosphatidyl choline trigger effects by binding to cell surface or nuclear receptors, altering mitochondrial permeability, or modifying critical intracellular proteins (314-316). The identification and synthesis of these relatively complex autoxidation products present a new

challenge and are likely to determine the pace at which this research can be conducted (313, 317, 318). Despite the intense effort expended over the past few years, our understanding of the mechanism and consequences of protein and DNA modification by reactive intermediatessboth oxidizing and alkylating agentssis still quite limited. The involvement of oxidative stress in a broad range of human diseases suggests that this is a fertile area for study with significant clinical potential. Acknowledgment. Work in L.J.M.’s laboratory was supported by NIH Grants CA87819 and ES013125. J.D.W. was supported by NIH Training Grant CA78136.

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