Pathways of cell signaling in hyperoxia

Free Radical Biology & Medicine, Vol. 35, No. 4, pp. 341–350, 2003 Copyright © 2003 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

doi:10.1016/S0891-5849(03)00279-X

Serial Review: Role of Reactive Oxygen and Nitrogen Species (ROS/RNS) in Lung Injury and Diseases Guest Editor: Brooke T. Mossman PATHWAYS OF CELL SIGNALING IN HYPEROXIA PATTY J. LEE*

and

AUGUSTINE M. K. CHOI†

*Section of Pulmonary and Critical Care Medicine, Yale University School of Medicine, New Haven, CT, USA; and †Division of Pulmonary, Allergy, and Critical Care, University of Pittsburgh Medical Center, Pittsburgh, PA, USA (Received 16 December 2002; Revised 9 April 2003; Accepted 17 April 2003)

Abstract—Administration of high concentrations of oxygen (hyperoxia) is a mainstay of supportive treatment for patients suffering from severe respiratory failure. However, hyperoxia, by generating excess systemic reactive oxygen species (ROS), can exacerbate organ failure by causing cellular injury. Therefore, a better understanding of the signal transduction pathways in hyperoxia may provide the basis for effective therapeutic interventions. The major biological effects of hyperoxia include cell death, induction of stress responses, inflammation, and modulation of cell growth. Major signaling pathways that appear to be involved include the mitogen-activated protein kinases (MAPKs), AP-1, and NF-␬B, which converge, ultimately, to the expression of a range of stress response genes, cytokines, and growth factors. © 2003 Elsevier Inc. Keywords—Hyperoxia, Oxidant injury, Reactive oxygen species, Signal transduction, Mitogen-activated protein kinases, Free radicals

INTRODUCTION

There appears to be significant overlap between the pathways activated in endothelial and epithelial cells during hyperoxia. Although simultaneous, parallel studies have not been performed in both cell types in a rigorous manner during hyperoxia, the major pathways implicated have been noted in a variety of cell types. Similar to findings in epithelial cells, endothelial cells exhibit growth arrest [1,2], ROS formation [3], and apoptosis [4,5] during hyperoxia. In addition, endothelial cells upregulate MAPKs [6], heat shock proteins [7], antioxidant enzymes [8,9], and adhesion molecules [10] in response to hyperoxia, which are also the major pathways implicated in epithelial cells during hyperoxia. We have unpublished data that, although both lung endothelial and epithelial cells activate MAPKs in response to 95% oxygen, the endothelial cells activate all three MAPKs (ERK, JNK, and p38) whereas the epithelial cells activate only ERK. Therefore, although differences in signaling patterns likely exist between cell types, this remains an underexplored area. Furthermore, the signaling and biological responses of fibroblasts and macrophages to hyperoxia have yielded significant insights into the effects of hyperoxia. This review will

Hyperoxia leads to the generation of reactive oxygen species (ROS), the collective term for superoxide anion (O2•⫺), hydrogen peroxide (H2O2), hypochlorous acid (HOCl), and the hydroxyl radical (OH•). The damaging effects of hyperoxia-generated ROS, although highlighted in the respiratory epithelium and endothelium in vivo, can cause systemic cellular and organ injury. Endothelial and epithelial cells are not only targets of hyperoxic injury, leading to the breakdown of critical epithelial-endothelial barriers and, ultimately, organ integrity, but also potential sources of ROS, which make them an intense focus for investigations.

This article is part of a series of reviews on “Role of Reactive Oxygen and Nitrogen Species (ROS/RNS) in Lung Injury and Diseases.” The full list of papers may be found on the homepage of the journal. Address correspondence to: Dr. Patty J. Lee, Yale University School of Medicine, Section of Pulmonary and Critical Care Medicine, 333 Cedar Street, P.O. Box 208057, New Haven, CT 06520-8057, USA; Tel: (203) 785-5877; Fax: (203) 785-3826; E-Mail: patty.lee@ yale.edu. 341

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Fig. 1. General schema for hyperoxia signaling in cells.

highlight the known signaling pathways in a variety of cells. Biochemical mechanisms of hyperoxia-mediated cell injury include lipid peroxidation, protein sulfhydryl oxidation, enzyme inactivation, DNA damage, and depletion of cellular reducing agents [11]. The effect of hyperoxia at the cellular and molecular levels is complex, as reflected in the multiple gene responses, variable regulation of hyperoxia-induced genes and proteins, and likely redundant signaling pathways that lead to pleiotropic biological effects. Little is known about the most proximal, or submembrane, component of oxygen signaling in cells. The generally accepted notion, which applies to bacteria, yeast, and mammals, is that heme proteins sense changes in oxygen tension with subsequent transfer of electrons along a signaling pathway that depends on reactive oxygen species [12]. These heme-based sensors include NAD(P)H oxidases, but direct evidence is lacking for hyperoxia-treated mammalian cells. Our laboratory has found that a chemical inhibitor of NAD(P)H attenuated hyperoxia-induced ROS in mouse epithelial cells, implicating the NAD(P)H oxidase system as a major source of ROS generation in these cells [13]. NADPH oxidase is a multicomponent enzyme complex present in the membranes of various cells. Functional assembly of the oxidase catalyzes the transfer of one electron from cytosolic NADPH to molecular oxygen, generating superoxide anion [14]. Although NADPH oxidase in neutrophils is known to play a critical role in host protection against infection, recent works show that virtually all cell types have a similar oxidase system. Furthermore, in a variety of nonphagocytic cells, NADPH oxidase-dependent O2⫺/H2O2 generation is observed in response to divergent extracellular stimuli [15]. Given that the generation of ROS, potentially through NADPH oxidase, is a well-established consequence of hyperoxia, with the ability to modulate multiple redox-

sensitive cellular functions, our simplified signaling schema starts with ROS (Fig. 1). One approach to consolidate our current understanding of hyperoxia is to start with the well-established biological effects of hyperoxia and then to delineate what is known about the signaling pathway(s) associated with the particular biological effect. Recognizing that many key processes overlap significantly, we can generally categorize hyperoxia-induced responses in mammalian endothelial cells into cell death, survival or stress responses, inflammation, and modulation of cell growth. CELL DEATH

A cardinal feature of hyperoxia in isolated cell culture systems and in vivo models is cell death [13,16 –19]. The issue of whether hyperoxia induces primarily an apoptotic or nonapoptotic death in vivo and in vitro is complex, as recently reviewed by Albertine and Plopper [20]. Suffice it to say that there are likely multiple death pathways occurring in response to oxygen toxicity, with features of both necrosis and apoptosis and the various assays used (including DNA laddering, TUNEL, and caspase activation), are not specific enough to reliably distinguish between the two modes of death. Apoptosis and necrosis are likely not mutually exclusive processes, especially in vivo in a complex organ such as the lung where more than 40 cell types exist. The in vivo and in vitro findings can be consolidated by the fact that at any one time during hyperoxia in the animal lung, for instance, certain cells or cell types undergo apoptosis while others undergo necrosis. The numerous cell types present in the lung, the local inflammatory milieu, and the presence or absence of local or circulating survival signals specific for certain cell types may all contribute to the presence of both apoptosis and necrosis in vivo. On the other hand, the in

Signal pathways in hyperoxia

vitro system reflects one cell type in the absence of a biological and inflammatory milieu, but is invaluable in trying to delineate the specific mechanisms and pathways involved in hyperoxia. Ultimately, both in vitro and in vivo systems studied in tandem will likely yield the most significant insights into the biology of hyperoxia. There is accumulating evidence that signaling pathways such as the mitogen-activated protein kinases (MAPKs), specifically the extracellular signal-regulated kinase or ERK1/2 MAPK, may have an important role in regulating hyperoxic cell death. MAPKs are a well-studied signal transduction system present in a wide variety of eukaryotes. MAPKs transduce signals for processes as diverse as mating, cell proliferation/differentiation, and cell survival/death. The major subfamily members include extracellular signalregulated kinase (ERK1/2), c-Jun N-terminal protein kinase (JNK1/2), and p38 kinase [21]. Each MAPK is activated through dual phosphorylation via a specific phosphorylation cascade. ERK1/2 is generally considered to be a survival mediator involved in the protective action of growth factors against cell death, but it has also been reported that the induction of hyperoxic cell death can be mediated via ERK1/2 in mouse macrophages [16]. There is recent data that p38, in conjunction with ERK1/2, may play a role in regulating hyperoxia-induced ROS generation [6], but the role of p38 in hyperoxia-induced cell death is as of yet unclear. Morse and colleagues showed that JNK1-deficient mice are more susceptible to hyperoxia and have increased lung epithelial cell apoptosis [22]. The observation that the MAPKs also activate activator protein-1 (AP-1) and nuclear factor ␬B (NF-␬B), key hyperoxia-induced transcription factors described later in this review, also lends support to the integral role MAPKs have in hyperoxia signaling. Recently, our laboratory showed that ERK1/2 MAPK is activated in murine lung epithelial cells [13]. Similarly, ERK1/2 MAPK activation after hyperoxia has been observed in murine macrophages and rat pheochromocytoma cells [16,23]. Buckley and colleagues showed that primary rat alveolar epithelial cells (AEC2) obtained from rats exposed to hyperoxia exhibited increased activation of ERK1/2 MAPK during the attachment or recovery phase in cell culture [24]. Furthermore, protection against hyperoxia-induced DNA breakage and apoptosis was conferred by ERK1/2 activation [24]. However, given that the ERK1/2 functions were studied in isolated hyperoxic rat type II cells recovered in room air, it is unclear whether ERK1/2 protects the cells from apoptosis during hyperoxia or during recovery. We showed that hyperoxia-induced murine lung epithelial cell death is attenuated by inhibiting ROS generation or by inhibiting ERK1/2 activation prior to hyperoxic exposure [13]. Therefore, the point at which ERK1/2 is inhibited (dur-

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ing initial hyperoxic exposure or recovery), cell type (primary vs. an immortalized line), and species (rat vs. mouse) may be important factors in the specific role of ERK1/2 in hyperoxia-induced cell death. Potential events upstream and downstream of ERK1/2 activation in hyperoxia-induced cell death include the modulation of Fas, p53, p21, Bcl, Bax, and caspases, which are all prototypic components of cell death/survival in various other injury models. Despite the observations of increased p53 and Fas levels after hyperoxia, null p53 and lpr mice (lacking functional Fas gene) do not exhibit increased cell death or lethality following hyperoxia [25,26]. Buckley and colleagues showed that hyperoxia induces p53, p21, and Bax proteins, suggestive of apoptosis, in AEC2 cells [27]. Similarly, Barrazone and colleagues showed that hyperoxia induces RNA and protein levels of Fas, p53, Bax, and Bcl-x in whole mouse lung but, interestingly, the “executioner” machinery for apoptosis, caspase 1 and 3 activities, was not significantly elevated [25]. The induction of Bcl-2 was also observed in transgenic IL-6 mice exposed to hyperoxia, raising the issue of a potentially important role of Bc1-2 family proteins in this model of cytoprotection [28]. However, there is also some conflicting data regarding the expression of Bcl-2 and Bax after hyperoxia in mouse lungs. Other investigators have found that, although mRNA levels of Bax were increased after 48 – 88 h of hyperoxia in mouse lung, there was little change in Bax protein and they did not observe Bcl-2 induction in mouse lung [26]. Given that ROS generation and many of the key death pathways focus on the mitochondria, including cytochrome c release, caspase activation, participation of Bcl-2 family members, and redox changes, there are surprising recent findings that mitochondrial generation of ROS is not required for hyperoxia-induced cell death at least in fibrosarcoma cells that lack mitochondrial DNA [29]. Notably, there are potentially other intracellular sources of ROS generation such as the membrane NADPH oxidase complex present in various cells. The authors do highlight, however, the important role of Bax or Bak in hyperoxia-induced apoptosis and the ability of Bcl-xl overexpression to prevent cell death. They postulate that the regulation of cell death following hyperoxia by Bcl-2 family members may be a potential mechanism whereby a variety of strategies protect against hyperoxia. Indirect evidence to support this notion is provided by Lu and colleagues, who did not investigate Bcl-2 function but demonstrated that the serine-threonine kinase Akt, which phosphorylates Bcl-2 family member Bad and thereby releases Bcl-xl, protects mice from lethal hyperoxia [30]. Finally, the roles of specific downstream death effectors, such as caspase 3 and PARP, remain to be eluci-

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dated in future studies. There has been evidence of cross-talk between MAPKs that appears to be important for hyperoxia-induced cell death and caspase activation [31]. Our laboratory has found that caspase 3 is activated in response to hyperoxia in mouse lung epithelial cells. Activation of caspase 3 was also confirmed by the cleavage of PARP, a major substrate of activated caspase 3 [13]. Although Barrazone and colleagues observed that at 24, 48, and 90 h hyperoxia caspase activities in whole lung lysates were not increased, at 72 h hyperoxia, which is the time point we examined, they found a 2-fold increase in caspase 3 activity [25]. SURVIVAL/STRESS RESPONSES

The stress response is one of the most highly conserved and adaptive aspects of all cells. Therefore, in the face of hyperoxic stress organisms have evolved sophisticated and redundant responses to ensure survival. The classic antioxidant defense system includes the “free radical detoxifying enzymes” such as the superoxide dismutases (SODs), glutathione peroxidase, and catalase. Indeed, the importance of the SODs in protecting against oxygen toxicity has been shown by the fact that homozygous SOD mutant mice have increased sensitivity to hyperoxia [32]. The corollary has also been found to be true; transgenic mice that overexpress either manganese SOD or copper-zinc SOD were more resistant to oxygen toxicity [33,34]. Other important antioxidant enzymes in hyperoxia include thioredoxin and peroxiredoxin. Thioredoxin is a protein disulfide oxidoreductase that was found to acutely increase at the transcriptional level in the lungs of newborn primates, implicating its role in the transition from the anaerobic intrauterine environment to relatively hyperoxic ambient air [35]. Additionally, studies from the same laboratory recently found that peroxiredoxin mRNA was induced by hyperoxia in primate lungs and involved protein kinase C signaling [36]. Heat shock proteins (HSPs) are a family of highly conserved, stress-inducible proteins that are ubiquitous, occurring in all organisms from bacteria and yeast to humans [37,38]. The principal HSPs range in molecular mass from ⬃15 to 110 kDa. The most well-studied and understood HSPs in mammals are those with molecular masses of ⬃60, 70, 90, and 110 kDa. Although HSPs were first described as a set of proteins whose expression was induced by heat stress, recent evidence suggest that a variety of cellular stresses including ischemia/reperfusion, hypoxia, energy depletion, and ROS formation can induce HSPs, in particular HSP70 [38,39]. Major physiological cellular stresses activate the inducible form of the 72 kDa heat shock protein (HSP70) expression. This induction of HSP70 is primarily via the transcription factor heat shock factors (HSFs), which when activated

bind to heat shock elements (HSE) in the promoter region of the HSP70 gene. Proposed mechanisms of cellular protection from HSPs include functioning as molecular chaperones to assist in the assembly and translocation of newly synthesized proteins within the cell, repair and refolding of damaged proteins, the maintenance of structural proteins, the prevention of protein aggregation, and the degradation of unstable proteins [40,41]. Interestingly, it has also been noted that HSPs can play a role in apoptosis. HSP27, HSP70, and HSP90 proteins are predominantly antiapoptotic, whereas HSP60 is proapoptotic [42– 44]. Moreover, it appears that these HSPs function at multiple points in the apoptotic signaling pathway to elicit these responses. Heme oxygenase-1 (HO-1), initially characterized as HSP32, has been found to have an important role in hyperoxic injury. HO-1 is an enzyme that degrades heme to bilirubin, is markedly induced by hyperoxia in multiple cell types including the lungs, and has been found to have profound protective effects. Overexpression of HO-1 in human epithelial cells conferred resistance to hyperoxia [45]. This effect was corroborated in vivo when exogenous HO-1 by gene transfer attenuated hyperoxic lung injury in rats [46]. One of the most interesting facets of HO-1 biology is that the reaction products of HO-1 catalysis, bilirubin, carbon monoxide, and ferritin, also have intrinsic antioxidant, anti-inflammatory, and antiapoptotic properties that can collaboratively impose significant cytoprotective effects. These topics have been thoroughly reviewed recently [47,48]. Intriguing roles for nonenzymatic antioxidants such as vitamins have also been investigated. Vitamins C and E were found to be protective in kidney epithelial cells exposed to hyperoxia [49]. Recently, vitamin E deficiency has been noted to predispose rat alveolar cells to apoptosis [50] and vitamin C prevented hyperoxia-induced apoptosis in human airway epithelial cells [51]. All of this points to a potential role for the protective effects of vitamins in hyperoxia-induced cell death. Retinoic acid may also prove to have an interesting role in hyperoxic injury given findings that retinoic acid protects against hyperoxia-mediated cell cycle arrest in lung alveolar epithelial cells by modulating cyclin activity [52]. INFLAMMATION

Hyperoxia has long been known to induce an inflammatory profile, as evidenced by increased TNF-␣, IL-1, neutrophil accumulation, and adhesion molecule expression. As reviewed by Barazzone and White, pretreatment with cytokines such as TNF, IL-1, and LPS induced tolerance to lethal hyperoxia presumably through the upregulation of the antioxidant responses such as manganese SOD [52a]. The role of TNF-␣ in hyperoxic

Signal pathways in hyperoxia

injury has been unclear. Studies using either TNF-␣ or antibodies to TNF-␣ showed increased survival in mice exposed to hyperoxia [53–55]. Pryhuber and colleagues, however, used mouse models of TNF-␣ receptor deficiency (targeted gene ablation of TNFR-I/p55 and TNFR-II/p75) to examine the role of TNF-␣ in hyperoxic lung injury [56]. They showed that hyperoxia-induced lung toxicity is in part mediated by TNFR-I. However, specific cytokine/chemokine gene induction and lethality were independent of TNFR-I and, therefore, therapeutic efforts to block TNF-␣ function may not be effective. It is also becoming clear that cytokine overexpression, in the absence of antioxidant enzyme upregulation, can afford significant protection against hyperoxia. For instance, transgenic mice that overexpress IL-6 exhibit markedly enhanced survival in hyperoxia compared to their transgene negative controls [28]. The protective effects were not associated with alterations in SOD activity but with increased Bcl-2 and tissue inhibitor of metalloproteinase-1 (TIMP-1) accumulation. Waxman and colleagues demonstrated that IL-11 transgenic mice also have improved survival in hyperoxia [57]. This was associated with minor changes in lung antioxidants but diminished hyperoxia-induced IL-1 and TNF production.

CELL GROWTH

Cellular growth arrest is a commonly observed phenomenon in hyperoxia. Of the various mechanisms that have been invoked, including the modulation of histone and thymidine kinase [58], defective S phase progression [59], and NF-␬B activation [60], the roles of p21, “growth arrest and DNA damage” GADD proteins, and, most recently, DNA repair proteins appear to be the most prominent. Hyperoxia damages DNA and cells respond by expressing genes that regulate cell cycle, DNA repair, and apoptosis. O’Reilly and colleagues demonstrated increased tumor suppressor gene p53 and expression of its downstream target p21 in lung epithelium following hyperoxia [61,62]. Despite the observation that p53deficient mice do not exhibit more toxicity to hyperoxia, O’Reilly and colleagues showed that hyperoxia was able to induce p21 independently of p53 and, in fact, p21deficient mice were more susceptible to hyperoxic injury [63]. Furthermore, hyperoxia had no effect on DNA replication in p21-deficient mice, suggesting that p21 may protect from hyperoxia, in part, by inhibiting DNA replication and allowing additional time for DNA repair. Hyperoxia also increased GADD45 and GADD153 expression in bronchiolar and alveolar epithelium [64]. GADD45 facilitates DNA repair via proliferating cell nuclear antigen and also synergizes with p21 to arrest

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cell growth. GADD153 also inhibits cell proliferation by inactivating C/EBP transcription factors [64]. The concept that growth arrest is an important defense against oxygen toxicity has been further highlighted by the recent demonstration that lung adenocarcinoma cells maintained in G1 phase during hyperoxia exhibited less DNA damage and cell death than their proliferating counterparts [65]. A potential explanation given was that there was more time allowed for base excision repair during G1 arrest. Wu and colleagues made a more specific link between DNA repair and protection from hyperoxia when they transduced lung epithelial cells with DNA base excision repair genes before exposure to hyperoxia [66]. Expression of either human enzyme 8-oxoguanine DNA glycosylase (hOgg1) or Escherichia coli enzyme formamidopyrimidine DNA glycosylase (Fpg) decreased DNA damage and increased cell survival in hyperoxia, which raises an intriguing approach to cellspecific protection from the toxic effects of hyperoxia. A growing area of interest is the role of growth factors, namely, keratinocyte growth factor (KGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor (IGF) in hyperoxia. KGF has been shown to promote alveolar epithelial hyperplasia in culture and in vivo, but, interestingly, intratracheal instillation of KGF reduced hyperoxic mortality in rats [67]. In mice treated with recombinant human KGF during hyperoxia, the protective effect was also observed in epithelium and endothelium but thought to be mediated by modulating p53, Bax, Bcl-x, and plasminogen activator inhibitor-1 (PAI-1) [68]. VEGF may play a notable role in hyperoxia, specifically as a survival factor in oxidant injury. Klekamp and colleagues showed that the high expression of VEGF mRNA in the distal airway epithelium of adult rats is markedly attenuated with hyperoxia [69]. This decline in VEGF correlated with increased TUNEL staining, and they postulated that the loss of VEGF contributes to the pathophysiology of oxygen-induced lung damage. IGF has been found by various groups to be significantly upregulated in hyperoxia, but the functional significance of this effect is as of yet unclear [70 –72]. Recent data supported the role of IGF-binding proteins in hyperoxia-induced apoptosis and growth arrest in lung epithelial cells [60]. TRANSCRIPTION FACTORS

AP-1 and NF-␬B are redox-sensitive transcription factors that prominently regulate hyperoxic responses. Both NF-␬B and AP-1 are activated in multiple cell types as well as in lung following hyperoxia [73–76]. NF-␬B activation has been mainly implicated in mediating the inflammatory responses such as IL-8 and TNF-␣ expression during hyperoxia. In certain studies,

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the downregulation of NF-␬B in hyperoxia has been associated with “favorable” effects. In human pulmonary artery endothelial cells, hyperoxia-induced activation of NF-␬B was abrogated by steroid treatment [77]. Steroids increased I␬B␣ expression during hyperoxia, thereby inhibiting NF-␬B activation, decreasing ICAM-1 expression, and, subsequently, decreasing neutrophil adhesion to the endothelium. Human bronchial and alveolar epithelial cells exhibited less cell injury in the presence of NF-␬B inhibition, potentially through increased glutathione levels [78]. However, recent data have also shown that NF-␬B activation can inhibit H2O2-induced alveolar epithelial cell apoptosis, while the suppression of NF-␬B activity augmented apoptosis [79]. Thus, the net effect of NF-␬B activation, whether deleterious or protective in hyperoxia, is likely complex due to the numerous genes regulated by NF-␬B. The functional roles of NF-␬B and AP-1 activities include the regulation of stress response genes. For instance, HO-1, manganese SOD, and glutathione peroxidase have NF-␬B or AP-1 binding sites in their 5⬘ regulatory regions. However, very little is known about the precise regulation of these genes by AP-1 or NF-␬B during hyperoxia. Aspects of antioxidant gene regulation in hyperoxia can be gleaned from the deletional and mutational analyses of the HO-1 gene in macrophages after hyperoxia. Lee and colleagues demonstrated that, in mouse macrophages, HO-1 transcriptional regulation is dependent upon both the “signal transducer and activator of transcription” (STAT) DNA-binding sites in the HO-1 promoter and AP-1 sites in the 5⬘ distal enhancer after hyperoxia [80]. Interestingly, the same HO-1 promoter and 5⬘ distal enhancer sites appear to be necessary and sufficient for hyperoxia-induced HO-1 gene transcription in rat endothelial cells, which invokes the potential importance of STAT and AP-1 transcription factors in additional cell types [80a]. The AP-1 transcription factors have been the focus of much attention in the oxidative stress arena due to the critical roles they play in the adaptive responses to stress as well as inflammation, cell death/survival, and differentiation/proliferation. A thorough review of AP-1 has been published recently by Otterbein and Choi [81]. A key highlight of their review is the importance of the extended AP-1 core sequence, or antioxidant response element (ARE), in regulating oxidative stress responses. The ARE sequence is present in many oxidant-responsive genes, including HO-1, NAD(P)H:quinone oxidoreductase (NQO1), and gluthathione S-transferase (GST), and may represent the mode whereby cells sense and respond to oxidative stress [81]. In addition to the classic AP-1 family members (Jun and Fos), other distinct transcription factors such as Maf and Nrf2 can bind to the ARE sequence [82– 84]. Nrf2 is a member of the

“cap and collar” family of bZIP transcription factors that is expressed in a wide variety of tissues including lung. Cho and colleagues demonstrated the physiological significance of Nrf2 when Nrf2 (-/-) null mice were found to be more susceptible to hyperoxic lung injury [85]. Intense investigations will be required to reveal additional members of the expanding AP-1 family and the intricate relationships of the various transcription factors in promoting oxidant-gene responses. CONCLUSION

The diverse pathways outlined in this review underscore the complex responses elicited by hyperoxia and the need for future studies to dissect the precise signaling mechanisms. Hyperoxia, by virtue of generating ROS, likely affects almost all cellular compartments and regulatory levels. For example, ROS can have direct effects on redox-responsive proteins that regulate key stress kinases and transcription factors, on signaling molecules such as p21, and on RNA translation and RNA/protein stability [12]. As summarized in Fig. 2, there are potentially key hyperoxia-induced signaling kinases, such as MAPK, PKC, or Akt, that modulate combinations of transcription factors such as members of the AP-1 or extended AP-1 family to elicit specific gene responses, which ultimately decide the fate of the cell or organism. Given the myriad of responses that can be generated from noxious stimuli such as hyperoxia, the cell uses an intricate and delicately balanced system involving multiple layers of signaling molecules and genes, thereby providing back-up modes. Based on our current knowledge, a potential scenario may include the coordinated roles of ROS activating ERK1/2 MAPK, then AP-1 or Nrf2, leading to transcription of stress response genes, and, ultimately, a cell that successfully resists hyperoxic damage. Simultaneously, additional regulatory circuits that may be activated are p21, GADD genes, and mobilization of DNA repair enzymes, leading to cell cycle arrest and repair of hyperoxic damage in the cell. Adding to the complexity of the system, the precise mode of cell death in hyperoxia is not yet well understood and the links between growth arrest and survival are only recently being uncovered. Furthermore, there appear to be age-related differences in tolerance to hyperoxia in many species, which invokes intriguing aspects of senescence and oxidative stress. Nevertheless, the extensive studies performed thus far do lend some insights into hyperoxia signaling and form a foundation for future strategies to abrogate injury. Although there are numerous candidate signaling pathways that may have some causal effects on hyperoxic injury, the pathways that decrease cell death (by modulating MAPKs, Akt, or Bcl-2 family proteins) or that

Signal pathways in hyperoxia

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Fig. 2. Summary of hyperoxia signaling pathways in cells. AP-1 ⫽ activator protein-1; GADD ⫽ growth arrest and DNA damage; HO-1 ⫽ heme oxygenase-1; ICAM ⫽ intracellular adhesion molecule; IGF ⫽ insulin-like growth factor; IL ⫽ interleukin; KGF ⫽ keratinocyte growth factor; MAPK ⫽ mitogen-activated protein kinase; NF␬B ⫽ nuclear factor ␬B; PARP ⫽ poly (ADP-ribosyl) polymerase; PKC ⫽ protein kinase C; ROS ⫽ reactive oxygen species; SOD ⫽ superoxide oxide dismutase; TNF-␣ ⫽ tumor necrosis factor ␣; VEGF ⫽ vascular endothelial cell growth factor.

enhance endogenous defense systems, such as antioxidants (SOD, glutathione, HO-1, vitamins) and growth factors (VEGF, KGF), appear to have significant potential as effective therapeutic targets.

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ABBREVIATIONS

AP-1—activator protein-1 ARE—antioxidant response element ERK— extracellular signal-regulated kinase JNK— c-Jun NH2-terminal kinase MAPK—mitogen-activated protein kinase MEK—MAP or ERK kinase NF-␬B—nuclear factor ␬B PARP—poly (ADP-ribosyl) polymerase p38 —protein 38 ROS—reactive oxygen species STAT—signal transducers and activators of transcription TUNEL—terminal deoxynucleotidyltransferase dUTP nick end labeling