Respiratory Physiology & Neurobiology 209 (2015) 106–114

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Effects of hyperoxic exposure on signal transduction pathways in the lung夽 Andrea Porzionato a , Maria Martina Sfriso a , Andrea Mazzatenta b , Veronica Macchi a , Raffaele De Caro a , Camillo Di Giulio b,∗ a b

Department of Molecular Medicine, University of Padova, Padova, Italy Department of Neurosciences and Imaging, University of Chieti, Chieti, Italy

a r t i c l e

i n f o

Article history: Accepted 1 December 2014 Available online 6 December 2014 Keywords: Reactive oxygen species Bronchopulmonary dysplasia Oxidative damage ERK JNK p38

a b s t r a c t Exposure to supraphysiological concentrations of oxygen is often applied in clinical practice to enhance oxygenation in acute or chronic lung injury. However, hyperoxic exposure is associated with increased reactive oxygen species production, which can be toxic to pulmonary endothelial and alveolar epithelial cells. Oxidative stress activates the pathways of the mitogen-activated protein kinases family: extracellular signal-regulated kinase (ERK1/2), C-Jun-terminal protein kinase (JNK1/2), and p38 kinase. Several studies have suggested that ERK activation in lung cells has a protective effect in response to hyperoxia, through stimulation of DNA repair and antioxidant mechanisms, and prolonged cell survival. Conversely, JNK1/2 and p38 kinase have been most frequently reported to have roles in induction of apoptotic responses. Moreover, exogenous factors, such as ATP, retinoic acid, substance P, thioredoxin, inosine and laminin, can have cytoprotective effects against hyperoxia-induced cell damage, through promotion of ERK activation and/or limiting JNK and p38 involvement. Published by Elsevier B.V.

1. Introduction Mechanical ventilation with supraphysiological concentrations of oxygen is often necessary to treat newborns, older children, and adults with respiratory distress due to hypoxemia, acute respiratory distress syndrome, or chronic obstructive pulmonary disease. However, in lung cells, hyperoxia can increase reactive oxygen species (ROS), which are extremely toxic and can cause cell injury and death (Xu et al., 2006). Hyperoxic lung damage is characterized by an extensive inflammatory response and damage to the alveolar–capillarity barrier, which can lead to impaired gas exchange and pulmonary edema. Such pathological changes in hyperoxic lungs are accompanied by injury and apoptotic or necrotic death of pulmonary cells (Mantell and Lee, 2000; Petrache et al., 1999). However, prolonged exposure to hyperoxia results in a scenario of both acute and chronic lung diseases, such as an acute inflammatory lung injury and bronchopulmonary

夽 This paper is part of a special issue entitled “Molecular basis of ventilatory disorders” guest-edited by Dr. Mietek Pokorski. ∗ Corresponding author. Tel.: +39 0871 3554044; fax: +39 0871 3554045. E-mail address: [email protected] (C. Di Giulio). http://dx.doi.org/10.1016/j.resp.2014.12.002 1569-9048/Published by Elsevier B.V.

dysplasia (BPD), respectively. In the acute inflammatory lung injury, NADPH oxidase activation generates ROS (e.g., superoxide anions, hydrogen peroxide, hydroxyl radicals, hypochlorous acid), which can directly injure pulmonary cells via lipid peroxidation, protein sulfhydryl oxidation, enzyme inactivation, DNA damage, and depletion of cellular reducing agents (Cacciuttolo et al., 1993; Zhang et al., 2003). The final effects in mammalian endothelial and epithelial cells are stress responses, and modulation of cell growth, inflammation, and/or death (Lee and Choi, 2003). BPD is the most common chronic lung disease of prematurity, and it results in impaired alveolar growth and a dysmorphic vascular architecture (Thebaud and Abman, 2007). In its classic form, BPD is strongly correlated to oxygen toxicity and mechanical injury. Moreover, most animal models of BPD involve hyperoxic exposure, such as with premature baboons, or newborn rats or mice in the early postnatal period. From a histopathological point of view, hyperoxia disrupts postnatal alveolar development, which leads to smaller numbers of enlarged and simplified alveoli, thicker septa, and an increase in alveolar macrophages (e.g., Dauger et al., 2003; Balasubramaniam et al., 2007; Porzionato et al., 2012a, 2013a; Grisafi et al., 2012, 2013). Experimental hyperoxic models of BPD also result in changes in

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microvascular development and thickening of the medial muscle layer of arteries, with pulmonary hypertension (e.g., Jones et al., 1984; Koppel et al., 1994; Porzionato et al., 2012a, 2013a; Grisafi et al., 2012, 2013). Moreover, hyperoxic exposure has also been reported to increase the number of lung mast cells, which preferentially accumulate around vessels (Brock and Di Giulio, 2006). Hyperoxic exposure and oxidative stress are known to activate many different cascades of intracellular signaling pathways. In particular, protein kinases, such as mitogen-activated protein kinases (MAPKs), might have roles in the production of the histopathological and functional changes that characterize hyperoxic lung damage. A thorough understanding into the regulation of the main signal transduction pathways in hyperoxia that can lead to alveolar epithelial cell injury and cell death might also provide the basis for effective therapeutic interventions. This review focuses on pulmonary cell responses to hyperoxia, with particular reference to the role of the main MAPKs.

2. Mitogen-activated protein kinases MAPKs are important intermediates in signal transduction pathways, and they have been conserved through evolution. To date, four different mammalian MAPK cascades have been described: extracellular signal-regulated kinase 1 and 2 (ERK 1/2); c-Jun N-terminal kinase (the JNK family); p38; and ERK5 (Plotnikov et al., 2011). Each cascade involves the three core kinases MAP3K, MAPKK, and MAPK, and usually also include additional upstream (MAP4K) and downstream components. These cascades can be activated by various extracellular stimuli, such as hyperoxia, and they control a wide range of fundamental cellular processes, such as cell growth, proliferation, differentiation, motility, stress responses, survival, and apoptosis (Lee and Choi, 2003; Plotnikov et al., 2011; Son et al., 2011; Zaher et al., 2007). Each MAPK carries out its function through dual specific phosphorylation. The signals transmitted need to be transported into the nucleus and to modulate specific intracellular and nuclear substrates that trigger the activities of transcription factors, transcription suppressors, and chromatin remodeling proteins in de novo gene expression (Plotnikov et al., 2011). Currently, some potential targets of the MAPK pathways include the family of proapoptotic caspases, cytokines (e.g., interleukin [IL]-6, IL-11 and IL-␤), growth factors, and heme oxygenase (HO-1). The downstream effectors of hyperoxia-induced activation of MAPKs are also redox transcription factors, such as nuclear factor-kappa ␤ (NF␬␤), AP-1 and NF-E2-related transcription factor 2 (Nrf2), which are involved in protective responses against oxidative damage (Zaher et al., 2007). Dysregulation or incorrect functioning of the MAPK pathways are involved in the progression of various diseases, such as cancer, diabetes, autoimmune diseases, and developmental abnormalities (Plotnikov et al., 2011). One of the main damaging stimuli that can mediate or induce activation of MAPK pathways is the production of ROS, such as superoxide, hydroxyl radicals, and hydrogen peroxide, following hyperoxic exposure (Petrache et al., 1999). Furthermore, ROS can induce oxidative modifications of signaling proteins, by altering the structure and function of the proteins through modification of critical amino-acid residues. Indeed, mimicking oxidative stress through direct exposure to exogenous H2 O2 leads to activation of MAPK signaling. MAPKs can also be activated by oxidative modifications to intracellular kinases; e.g., MAP3Ks that are involved in MAPK signaling cascades. Another potential mechanism for MAPK activation by ROS is the inactivation and degradation of MAPK phosphatases, which are tyrosine and serine/threonine phosphatases that are involved in negative

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regulation of MAPKs. Other factors that activate MAPK pathways are environmental stress and growth factors (Son et al., 2011). 2.1. ERK The MAPK/ERK pathway is also known as the Ras–Raf–MEK–ERK pathway, and it is a kinase cascade that has a central role in the signaling of a wide variety of extracellular agents that operate through different receptors-tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptors. Growth factor receptors are most commonly activated by ligand-induced dimerization or oligomerization, which phosphorylates RTKs. For example, insulinlike growth factor-1 (IGF-1) contributes to the activation of the ERK pathway via phosphorylation of EGF receptors (Son et al., 2011). In most cases, the activation of these receptors is transmitted to the small GTPase Ras by several mechanisms, and this in turn activates Raf, a MAP3K. Thereafter, the signal is transmitted to the MAPKKs MEK1 and MEK2, which, again in turn, transmit their signals to ERK1/2. Ligand-independent clustering and activation of growth factor receptors can be induced directly by exposure to different oxidative-stress-inducing agents, such as osmotic stress and ultraviolet radiation (Meves et al., 2001). The first step is the activation of cell-surface receptors through chemical cross-linking or aggregation of receptors and membrane rafts, which leads to the production of ROS, as second messengers of this intracellular signal transduction. The second step involves chemical modifications to protein tyrosine kinases, to initiate the tyrosinephosphorylation–dependent local switch for activation of the catalytic activity of the enzymes (Nakashima et al., 2005). Following this activation pathway, oxidative stress induces EGF receptor activation through RTK phosphorylation, and H2 O2 has been proposed to be a mediator of ligand-independent phosphorylation of growth factor receptors in response to oxidative stress (Meves et al., 2001). Phosphorylation of ERK1/2 supports the activation of hundreds of substrates in many cellular locations, including the cytoplasm, mitochondria, Golgi complex, endosomes, cellular membranes, and in particular, the nucleus. The final effect of activation of the ERK cascade is the induction of several processes, which include induction or suppression of transcription and chromatin remodeling, which mainly promotes cell proliferation and differentiation, but also cell apoptosis under some stress response conditions. The ERK cascade is generally considered to be a survival mediator that is involved in the protective actions of growth factors against cell death. For its role under hyperoxic conditions, most studies have reported pro-survival effects (Ahmad et al., 2004; Buckley et al., 1999, 2005; Carnesecchi et al., 2009; Chen et al., 2010; Kannan et al., 2006; Jones and Agani, 2003; Lang et al., 2010; Li et al., 2006, 2011a; Monick et al., 2004; Papaiahgari et al., 2004; Parinandi et al., 2003; Truong et al., 2004; Xu et al., 2006; Waldow et al., 2008), although under some conditions, pro-apoptotic actions have also been reported (Petrache et al., 1999; Zhang et al., 2003). Various studies have reported increased activation of ERK in response to hyperoxia, with this phenomenon frequently ascribing to cellular ROS generation (e.g., Kannan et al., 2006; Kim et al., 2012; Monick et al., 2004; Truong et al., 2004; Xu et al., 2006; Zhang et al., 2003). The role of the ERK pathway in hyperoxic exposure is illustrated in Fig. 1. Some of the mechanisms involved in ERK activation in response to hyperoxia and ROS generation will be discussed here. Mitochondria are the major source of ROS production under normoxic and hyperoxic conditions in which the ERK pathway is involved. In the mouse lung epithelial cells (MLE-12), ROS generation due to hyperoxic exposure results in disruption of both

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O2 AngII EXOGENOUS STIMULATORS ATP RA Inosine Trx Laminin

ROS

NADPH oxidase

A1TR

Nox1

DNA damage

CHI3L

mtALDH

ERK

Survival genes macrophages MLE

Necrosis/Apoptosis

Egr1 A1TR Nrf2 AP1

Necrosis/Apoptosis

Fig. 1. Schematic diagram of the ERK signaling pathway in lung cells exposed to hyperoxia. Elevated concentrations of oxygen can lead to reactive oxygen species (ROS) production, also via NADPH oxidase activation, which might be stimulated by angiotensinII (AngII). ROS promotes stimulation of mitochondrial aldeyde dehydrogenase (mtALDH) and chitinase 3-like 1 (CHI3L), and also causes DNA damage, which induces ERK activation. Exogenous factors, such as ATP, retinoic acid (RA), inosine, thioredoxin, and laminin, can induce ERK activation. Activated ERK leads to transcription of survival genes, like early growth response gene-1 (Egr-1), Ang-II type 1 receptor (A1TR), Nrf2, and activator protein 1 (AP1)-transcriptor factor (Jun), which inhibit necrotic and apoptotic cell death. In contrast, ERK activation might induce apoptosis of pulmonary macrophages.

the inner and outer mitochondrial membranes, which induces the loss of mitochondrial activity, such as a reduction in aldehyde dehydrogenase (mtALDH) activity, and cytoplasm vacuolization (Romashko et al., 2003; Xu et al., 2006). In particular, the expression of mtALDH is down-regulated in the newborn rat lung after hyperoxic exposure. Conversely, overexpression of mtALDH in A549 cells decreases the levels of intracellular and mitochondrion-derived ROS in response to hyperoxia, and reduces hyperoxia-induced cell death, thus having a protective role. These actions appear to be mediated by the ERK pathway, as mtALDH overexpression also stimulates ERK phosphorylation under normoxic and hyperoxic conditions, and inhibition of ERK phosphorylation partially suppresses the protective effects of mtALDH in hyperoxia-induced cell death (Xu et al., 2006). Numerous studies have shown that elevated levels of ROS generated during hyperoxia can lead to DNA damage. Many different DNA glycosylases have been identified as participating in the removal of oxidative base damage; first by recognition of the altered DNA bases, and then by catalysis of their hydrolytic excision. One of the most common oxidative adducts is 8-Oxo-2 -deoxyguanosine (8oxo-dG), which is mainly removed by human 8-oxoguanine DNA glycosylase (hOgg1). Kannan et al. (2006) revealed that in AEC2 and A459 cells, hyperoxic exposure increases the levels of 8-oxodG and enhances the activity of ERK. Furthermore, transfection of AEC2 and A459 cells to overexpress the DNA glycosylase hOgg1 resulted in a further increase in ERK activation that has been associated with decreased 8-oxo-dG levels and increased cell survival and proliferation. Other in vitro studies, instead, have demonstrated increased death of A459 cells exposed to a hyperoxic environment (95% oxygen), which was mainly through necrotic mechanisms. Here,

hyperoxia induces enhanced ERK signaling and diminishes Akt activation and protein expression, which has been proposed to be a pro-survival factor (Monick et al., 2004; Truong et al., 2004). Blocking upstream ERK activity leads to further increased cell death under hyperoxic conditions, which suggests that ERK activation has a role in limiting hyperoxia-induced necrotic cell death also due to reduced Akt activation and expression. In this way, ERK activity would compensate for the loss of Akt survival effects (Truong et al., 2004). Further studies focused on the Akt/ERK interaction have confirmed their active protective role against hyperoxic damage. For example, high serum chitinase-3-like 1 (CHI3L1; also known as YKL-40), which is a member of the evolutionarily conserved glycosyl hydrolase 18 family, has been linked to higher incidence of asthma, chronic obstructive pulmonary disease, and lung cancer (Kim et al., 2012). CHI3L1 and its mouse homolog BRP-39 have also been shown to be critical regulators of oxidant injury, inflammation, and epithelial-cell apoptosis in murine and human lungs (Sohn et al., 2010). In human airway epithelial cells, hyperoxia significantly enhances apoptosis and CHI3L1 expression, together with phosphorylation of ERK1/2 and p38, and with reduced Akt phosphorylation. Conversely, CHI3L1 knock-down protects cells from hyperoxia-induced apoptosis through increased phosphorylation of ERK1/2 (Kim et al., 2012). Supraphysiological concentrations of oxygen can also result in an extensive damage to the alveolar–capillary barrier, which increases in permeability. The vascular network of the lung is extremely vulnerable to oxygen toxicity, and in particular, endothelial cells are one of the main targets of a hyperoxic insult, which can result in extensive vasculature leaking (Papaiahgari et al., 2004; Parinandi et al., 2003). Hyperoxic exposure in the newborn period has been shown to decrease microvessel density and vascular endothelial growth factor (VEGF) expression in rat lung (e.g., Hosford and Olson, 2003; Remesal et al., 2009; Porzionato et al., 2012a, 2013a; Grisafi et al., 2012, 2013). Human pulmonary artery endothelial cells exposed to hyperoxia have been shown to undergo increased release and extracellular accumulation of O2 − and H2 O2 , due to stimulation of NADPH oxidase activity (Parinandi et al., 2003). Blocking NADPH oxidase with diphenyleneiodonium during hyperoxia significantly inhibits the generation of both intracellular and extracellular ROS, which indicates an active role of NAPDH oxidase for the conversion of oxygen into O2 − (Papaiahgari et al., 2004). ROS generation via NADPH oxidase induction appears to contribute to the triggering of the ERK pathway after exposure to high concentrations of oxygen (Papaiahgari et al., 2004; Parinandi et al., 2003). Further studies in mice have specifically identified NOX1 as the member of the NAPDH oxidase family that is mainly involved in activation of the ERK pathway (Carnesecchi et al., 2009). Thus, activation of the ERK pathway might be due to ROS generation by NADPH oxidases, or to a direct interaction with NOX1 (Carnesecchi et al., 2009). Hyperoxic exposure has been reported to increase ATP release in human lung endothelial cells, and this phenomenon appears also to be involved in ERK activation (Ahmad et al., 2004). Indeed, incubation of cells with ATP increases the activity of ERK, which suggests a role for ATP as an additional activator of the ERK pathway, at least in microvascular lung cells. Also in this setting, the outcome of ERK activation results in a survival cell response, which prevents hyperoxia-induced cell-cycle arrest (Ahmad et al., 2004). Apart from the impairment of alveolarization, hyperoxic exposure of neonatal rats is known to cause patchy areas of interstitial thickening (e.g., Porzionato et al., 2012a, 2013a; Grisafi et al., 2012, 2013) and to increase lung type I collagen and ␣-SMA levels, which suggests myofibroblast accumulation (Lang et al., 2010). These data were also confirmed by Li et al. (2011b), who demonstrated that hyperoxia can induce trans-differentiation of alveolar interstitial

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fibroblasts to myofibroblasts (AIF-to-MYF). Moreover, there also is an increase of angiotensin-II (Ang-II) which has been identified as a pro-fibrotic factor in experimental lung fibrosis. The expression of Ang-II type 1 receptor (AT1R) mRNA and protein and ERK phosphorylation are enhanced under these environmental conditions. Inhibition of ERK results in diminished expression of both type I collagen and ␣-SMA, without interfering with AT1R upregulation in hyperoxia. In contrast, inhibition of AT1R by AT1R small-interfering (si)RNAs promotes decreased expression of activated ERK, type I collagen, and ␣-SMA, which suggests that AT1R is an upstream activator of ERK, which in turn has a role in stimulation of the expression of type I collagen and the accumulation of myofibroblasts (Lang et al., 2010). Many different exogenous factors have been reported to modulate survival to hyperoxic stimuli through activation of the ERK signaling pathway. In vitro cell viability assays have revealed that pretreatment of AEC2 cells with thioredoxin significantly decreases hyperoxia-induced cell death and markedly reduces the generation of ROS induced by hyperoxia, as measured by flow cytometry. Moreover, both hyperoxia and thioredoxin induce ERK phosphorylation, although pretreatment of cells with thioredoxin provides 3-fold greater stimulation of ERK than for hyperoxic exposure alone, which will further contribute to the protective activation of ERK (Chen et al., 2010). Hyperoxic exposure is known to increase the number of TUNELpositive cells and to reduce cell proliferation in rat lungs (e.g., Li et al., 2006). Cultured AEC2 cells also undergo increased TUNEL positivity in response to hyperoxic exposure (Buckley et al., 2005). In vivo administration of retinoic acid in hyperoxia has been reported to markedly increase the number of proliferative cells and to significantly decrease apoptosis, although in the absence of evident effects on alveolarization (Li et al., 2006). Apart from these effects, retinoic acid has been shown to further up-regulate ERK signaling both in vivo (Li et al., 2006) and in cultured lung fibroblasts (Li et al., 2011a). Pretreatment of rats and cells with inosine also results in reduction of hyperoxia-induced DNA damage, with concomitant increase in ERK signaling activation. Conversely, upstream inhibition of ERK in hyperoxic rats pretreated with inosine shows an apoptotic pattern similar to hyperoxic untreated cells. In this way, inosine has been suggested to have a protective role through upstream signaling pathways that converge on ERK activation (Buckley et al., 2005). Previous studies have also demonstrated that significant protection against hyperoxia injuries can be conferred by laminin–epithelial cell interactions. Laminin is a component of the alveolar basement membrane, and it is a potential source of protective signaling. Buckley et al. (1999) showed that AEC2 cells in culture undergo DNA damage and apoptosis after 48 h of exposure to 95% oxygen. Culture of cells on laminin substrates, with respect to other plastic supports, results in increased activation of ERK and decreased death of AEC2 cells, which suggests a protective role of ERK in response to hyperoxia that is mediated by laminin (Buckley et al., 1999). To function against the damaging effects of hyperoxia, ERK modulates the expression of various genes. For instance, the expression of early growth response gene (Egr-1) is rapidly increased in stress responses. Jones and Agani (2003) demonstrated increased mRNA and protein levels of Egr-1 in both mouse alveolar carcinoma cells and lungs of newborn mice exposed to hyperoxia for prolonged periods. Inhibition of ERK completely blocked the hyperoxia-related induction of Egr-1, which further supports the role for the ERK pathway in the up-regulation of Egr-1 (Jones and Agani, 2003). Activated ERK promotes the induction of various downstream transcription factors, such as Nrf2. Under stress conditions, the

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nuclear translocation of Nrf2 results in activation of gene expression mediated by antioxidant response elements, which are involved in cellular protection against toxic insults. Indeed, inhibition of the ERK pathway blocks hyperoxia-enhanced Nrf2 nuclear accumulation and antioxidant-response-element–driven reporter expression in alveolar epithelial cells, which increases cell death (Papaiahgari et al., 2004). As well as the above survival effects of ERK in hyperoxia-exposed cells, other studies have reported apoptotic effects under other conditions. For instance, Petrache et al. (1999) demonstrated that ERK signaling has a role in alveolar macrophage apoptosis after hyperoxic exposure (95% oxygen). In futher detail, in vitro inhibition of the ERK pathway showed attenuation of apoptosis in cultured macrophages after hyperoxic insults. A marked in vivo increase in TUNEL-positive cells that resembled macrophages was shown in the alveoli of lungs from mice exposed to hyperoxia, compared to mice exposed to normoxia (Petrache et al., 1999). This proapoptotic action of ERK can be ascribed to the specific cell type involved (macrophages), which was not considered in the previously discussed literature, which mainly reported pro-survival effects. Likewise, Western blotting of MLE cells exposed to hyperoxia for up to 72 h has shown that ROS activates MAPKs, and in particular ERK1/2, which results in cell death. The upstream inhibition of ROS before hyperoxic exposure can block this induction of ERK (Zhang et al., 2003). Moreover, ERK inhibition with PD-98059 results in attenuation of hyperoxia-induced cell death, both in vitro and in vivo, which suggests an active role of this pathway in the induction of cell death (Zhang et al., 2003). Thus, ERK1/2 might have a dual roles in hyperoxia, as either promoters or inhibitors of cell death, and these roles appear to be dependent on the cell type and culture conditions (Zaher et al., 2007). 2.2. JNK The c-Jun NH2 -terminal kinases (JNKs) are also known as stressactivated protein kinases, and these are serine/threonine peptides that are encoded by three genes: Jnk1, Jnk2 and Jnk3. Jnk1 and Jnk2 are ubiquitously expressed, while Jnk3 has a more limited pattern of expression, as it is largely restricted to the brain, the heart and the testis (Davis, 2000; Huang et al., 2009; Makena et al., 2011). Stress and other stimuli transmit their signals to small GTPases, such as CDC42 and Rac1, which activate MAP3K kinases. In turn, MAP3Ks phosphorylate and activate MAPKK kinases (MKK4 and MKK7), finally stimulating JNKs. Upon stimulation, JNK phosphorylates many cytoplasmic and nuclear substrates, the activation of which promotes transcription of many genes involved in apoptosis, and immune and stress responses (Plotnikov et al., 2011). A major target of the JNK signaling pathway is the AP-1 complex; this complex is mainly composed of the transcription factors Jun and Fos, which are induced by JNK (Joseph et al., 2008; Li et al., 2003; Romashko et al., 2003). The JNK pathway has been implicated in the response to hyperoxia in various in vivo and in vitro models (Carnesecchi et al., 2009; Chen et al., 2010; Huang et al., 2009; Joseph et al., 2008; Li et al., 2003, 2011b; Makena et al., 2011; Romashko et al., 2003). Depending on the stimuli and the strength and duration of JNK activation, the cellular responses can be different, and can range from induction of apoptosis, to increased survival, and to proliferation changes (Wagner and Nebreda, 2009). The role of JNK signaling in hyperoxic exposure is illustrated in Fig. 2. Hyperoxia exposure (95% oxygen) of various cell types (e.g., rat lung fibroblasts, and A549, MLE, and AEC2 cells) has been reported to increase cell death (Buckley et al., 1999, 2005; Chen et al., 2010; Huang et al., 2009; Kannan et al., 2006; Li et al., 2003, 2011a,b;

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O2

EXOGENOUS FACTORS

ROS

NADPH oxidase Nox1

RA Substance P Trx

JNK

Fas

AIF-to-MYF differentiation

LC3B

Autophagy

TGFβ1 AP1 IL8 (c-Jun) Impaired alveolarization

Survival Inflammation Necrosis/Apoptosis Fig. 2. Schematic diagram of the JNK signaling pathway in lung cells exposed to hyperoxia. Hyperoxia leads to ROS production, also via NADPH oxidase activation. ROS stimulate the activation of JNK, which promotes alveolar interstitial fibroblasts to myofibroblasts (AIF-to-MYF) transdifferentiation. JNK promotes inflammation and impaired alveolarization through induction of AP-1 and transforming growth factor-beta1 (TGF␤1), respectively. Thus, the final effect of JNK activation supports necrosis and apoptosis. Exogenous factors, such as retinoic acid (RA), substance P and thioredoxin, inhibit JNK induction. JNK might stimulate microtubule-associated protein 1 light chain 3B (LC3B) factor, which would induce autophagic mechanisms to protect the cell against hyperoxia injury.

Monick et al., 2004; Romashko et al., 2003; Truong et al., 2004; Xu et al., 2006) and to increase the levels of total and phosphorylated JNK (Chen et al., 2010; Huang et al., 2009; Joseph et al., 2008; Li et al., 2003, 2011a,b; Romashko et al., 2003). However, a further study did not find any activation of the JNK pathways in MLE cells exposed to hyperoxia (Zhang et al., 2003). Conversely, inactivation of JNK by dominant-negative mutations or by a specific inhibitor can result in increased cell survival after hyperoxic exposure (Li et al., 2003; Romashko et al., 2003). As described above (Section 2.1), hyperoxia leads to ROS generation through the activation of NAPDH oxidase. This enzyme is also involved in JNK signaling. In particular, the NAPDH oxidase isoform NOX1 (but not NOX2) participates in hyperoxia-induced lung injury through phosphorylation of JNK. In parallel, NOX1 deficiency decreases JNK phosphorylation, which results in decreased cell death. Thus, also in this case, decreased phosphorylation of JNK is associated with protection against cell death (Carnesecchi et al., 2009). Another mediator that is implicated in hyperoxia-induced cell death and impaired alveolarization in the developing lung is transforming growth factor-beta 1 (TGF-␤1), which has also been recently implicated in the JNK pathway in the developing lung. Exposure of A459 and MLE cells to varying levels of hyperoxia (40%, 60%, 95% oxygen) increases the expression of TGF-␤1 and phosphorylation of JNK in a dose-dependent manner, which results in cell death. In this experimental condition, with inhibition of the JNK pathway, lung epithelial cells have been shown to have a higher percentage of survival. In vivo, instead, it has demonstrated that transgenic TGF-␤1 mice show significantly increased mortality under hyperoxia, compared to wild-type controls. Moreover, the use of a JNK inhibitor significantly increased survival and improved

alveolarization in transgenic TGF-␤1 mice under hyperoxia (Li et al., 2011b). Increased expression of Fos and Jun (Li et al., 2003) and increased levels of the phosphorylated c-Jun protein (Romashko et al., 2003) have been reported after hyperoxic exposure of wild-type MLE12 cells. In these cases, cells transfected with a dominant-negative Jun showed improved survival (Romashko et al., 2003). Several studies have demonstrated that AP-1 is activated in oxidant signaling, oncogenic transformation, immune responses, and cell differentiation and apoptosis (Li et al., 2003; Romashko et al., 2003; Joseph et al., 2008). Indeed, MLE-12 cells transfected with AP-1 reporter constructs saw significantly enhanced gene expression of the AP-1 reporter under hyperoxic conditions, which indicates that AP-1 is activated by hyperoxia (Romashko et al., 2003). A series of factors have also been shown to have protective roles in hyperoxia-exposed cells, through their inhibition of the JNK pathway. For instance, the addition of substance P to cell cultures decreases JNK expression, decreases hyperoxiainduced apoptosis, and increases cell proliferation (Huang et al., 2009). Under hyperoxic conditions, administration of the antioxidant thioredoxin in cultured AEC2 cells also significantly inhibited JNK phosphorylation, protected the cells from apoptosis, and increased cell proliferation (Chen et al., 2010). Retinoic acid also has a protective role in hyperoxia-induced cell injury, through down-regulation of JNK in rat lung fibroblasts (Li et al., 2011a). ROS production and activation of the JNK pathway has been suggested to have a role in inflammatory responses to hyperoxia. High oxygen concentrations stimulate cytokine production, including tumor necrosis factor-␣, IL-1␤, IL-6, and IL-8, (Otterbein et al., 2003). In cultured epithelial lung cells exposed to hyperoxia, increased IL-8 was reported to be correlated with JNK activation and increased AP-1 activity. Moreover, it has been reported that JNK inactivation or ROS inhibition mediated by superoxide dismutase can markedly reduce this inflammatory cytokine production (Joseph et al., 2008). The JNK pathway appears to also have a role in the hyperoxia-induced AIF-to-MYF transition, as concomitant inhibition of JNK blocks this transition in A459 cells exposed to hyperoxia (Li et al., 2011b). As well as apoptotic and necrotic mechanisms induced by hyperoxic conditions, autophagy is a cellular homeostatic process that is responsible for lysosomal turnover of organelles and proteins. Autophagy has also been implicated in the general response to oxidative stress of cells and tissues. Exposure of mice to hyperoxia (>95% oxygen, 72 h) activates morphological and biochemical markers of autophagy, such as increased expression of microtubule-associated protein 1 light chain 3B (LC3B). The expression and activation of LC3B is mainly promoted by hyperoxia-induced increases in cellular ROS, through activation of the JNK pathway. It is worth noting that overexpression of LC3B confers cytoprotection after hyperoxia, by inhibition of the cleavage of caspase-3, and initiation of apoptosis (Tanaka et al., 2012). Jnk1−/− mice continuously exposed to >98% oxygen show increased pulmonary epithelial TUNEL-positive cells associated with increased mortality. Jnk2−/− mice have demonstrated similar, but less pronounced, susceptibility to hyperoxia. Simultaneous deletion of both Jnk1 and Jnk2 cannot be examined in vivo, due to the embryonic lethality of this condition in mice. These results have suggested that the JNK1/2 pathways participate in the protective responses to oxidative injury, and that these two genes cooperate with each other to produce physiological compensation (Morse et al., 2003). These data thus suggest a positive role for JNKs in cell survival, although most studies propose JNK as a pro-apoptotic factor.

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O2 CO

ROS

EXOGENOUS FACTORS RA Substance P Trx

hOgg1

PKCδ

CHI3L

p38 HO-1 Necrosis/Apoptosis Survival Fig. 3. Schematic diagram of the p38 signaling pathway in lung cells exposed to hyperoxia. Elevated concentrations of oxygen lead to ROS production, which leads to direct activation of p38. P38 can be induced via protein kinase C-␦ (PKC␦) and CHI3L activation. Activated p38 supports necrosis and apoptosis. Moreover, exogenous factors, such as retinoic acid (RA), substance P, carbon monoxide (CO) and thioredoxin, as well as activation of DNA repair signaling (hOgg1), might inhibit p38.

2.3. P38 P38 is another member of the MAPK family that is activated by stress stimuli and signals that are transmitted through the recruitment of different receptors. The signals of the p38 cascade are transmitted via small GTPases, MAP4K and MAP3K, which are usually involved in the JNK signal pathway. In contrast to the JNK pathway, MKK3 and MKK6 are involved instead of MKK4 and MKK7. Apart from its role in stress responses, p38 has a central role in regulation of immunological effects, cell apoptosis, senescence, and survival, and cell-cycle checkpoints (Plotnikov et al., 2011), as p38null mice die as embryos (Otterbein et al., 2003). The role of p38 in response to hyperoxia-induced damage in the lung is as yet unclear, as the effects of its activation have given controversial results. Some studies have reported prosurvival effects of p38 activation in response to hyperoxia, while other studies have mainly observed detrimental actions. The pathway of p38 signaling in hyperoxic exposure is illustrated in Fig. 3. In terms of the upstream regulatory elements of p38 activation, Otterbein et al. (2003) observed that MKK3−/− mice were hypersusceptible to hyperoxic exposure, as they died after a few days of hyperoxic exposure. Otterbein et al. (2003) also suggested a possible involvement of p38 activation in the beneficial effects of carbon monoxide (CO) on hyperoxia-induced lung damage. Treatment with exogenous administration of CO limits the development of hyperoxia-associated histopathological changes and attenuates hyperoxia-induced cytokine expression. Conversely, the in vitro administration of CO in A459 cells exposed to hyperoxia and transfected with p38 dominant-negative constructs did not have any cytoprotective effects, which indicated that the action of CO is mainly due to the activation of p38 signaling (Otterbein et al., 2003). P38 activation has also been proposed to protect from hyperoxic damage through an increased expression of HO-1. Indeed, a previous study reported that hyperoxia induced activation of the stress protein HO-1, which promoted growth arrest in A549 epithelial cells and conferred protection against hyperoxia (Lee et al., 1996). HO-1 has been reported to be partly located in plasmalemma caveolae of endothelial and epithelial pulmonary cells, and in oxidative

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lung injury, it confers cytoprotection from anti-inflammatory, antiapoptotic and anti-proliferative effects. In particular, caveolin-1 (Cav-1) has been reported to participate in vesicular trafficking and in signal transduction events such as apoptosis. In vitro, Cav1 knock-out pulmonary fibroblasts, lung endothelial cells, and bronchial epithelial cells exposed to 95% oxygen are resistant to hyperoxia-induced cell death approximately three times more than the respective wild type cells. In these transgenic cells, increased phosphorylation of p38 and elevated accumulation of HO-1 have been observed. These data suggested that hyperoxic exposure of Cav-1−/− mice enhances p38 activation, which induces HO-1 protection of cells against hyperoxic insult (Jin et al., 2008). Other studies, instead, have focused on the involvement of caveolae in the p38 signaling pathway, as numerous GTPases have been localized to caveolae (Lee et al., 1996; Jin et al., 2008). As well as the above studies that indicate a pro-survival role of the p38 pathway, other studies have reported detrimental effects after the activation of the p38 pathway in hyperoxia. For example, in terms of ROS production, hyperoxic exposure of human pulmonary artery endothelial cells induces a significant increase in intracellular ROS, which results in phosphorylation of both ERK and p38. Transfection of cells with p38 dominant-negative constructs, in turn, significantly attenuates the generation of ROS under hyperoxic exposure (Parinandi et al., 2003). P38 has been reported to have an apoptotic role in hyperoxia in AEC2 cells (Chen et al., 2010) and lung fibroblasts (Li et al., 2006, 2011a). Under hyperoxic conditions, administration of the antioxidant thioredoxin to cultured AEC2 cells significantly inhibited p38 phosphorylation and protected the cells from apoptosis (Chen et al., 2010). The same protective effect took place in lung fibroblasts treated with retinoic acid, which down-regulated p38 activity, decreased apoptosis, and stimulated cell proliferation (Li et al., 2006, 2011a). To confirm the protective role of the administration of substance P during hyperoxia, further studies have shown that TUNELpositive nuclei increase markedly in the lungs from rats exposed to hyperoxia, and decrease significantly after substance P treatment. Substance P significantly suppresses the activity of NADPH oxidase and reduces ROS production, which results in a block of the p38 signaling pathway (Huang et al., 2015). P38 has also been reported to be activated with the JNK pathway in a synergistic manner. Indeed, MLE-12 cells exposed to hyperoxia have shown enhanced activation of the p38 and JNK proteins, together with increased cell death. The selective inhibition of p38 is sufficient to increase cell survival under hyperoxic conditions (Romashko et al., 2003). Considering hyperoxia-induced DNA damage, the p38 cascade is also involved. Molecular analysis has shown that hyperoxia induces a 7-fold increase in p38 activity in A459 cells compared to controls, while p38 dominant-negative transfected cells present a reduction of apoptosis under hyperoxic exposure. Moreover, overexpression of hOgg1 significantly decreases p38 activity under oxygen treatment, which correlates with a decrease in hyperoxic damage (Kannan et al., 2006). As described above, the p38 pathway is involved in the CHI3L1 signaling in chronic obstructive pulmonary disease (Kim et al., 2012). It has been shown that hyperoxia enhances CHI3L1 production and phosporylation of p38, which promotes apoptotic cell death. In contrast, p38 activation decreases in CHI3L1deficient cells, which survive longer than their controls (Kim et al., 2012). The involvement of the p38 cascade has been observed in cultured LMVEC endothelial pulmonary cells, which showed increased caspase-3 activation and DNA laddering in response to hyperoxia (Grinnell et al., 2012). In particular, the higher incidence of ROS-induced apoptosis was correlated with significantly increased

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activation of p38 and with increased production of the caspaseinduced protein kinase C␦ (PKC␦) cleavage products. PKC␦ has been reported to be activated by oxidative stress, and it is an important modulator of apoptosis. Preincubation of LMVECs with a PKC␦ chemical inhibitor significantly attenuated p38 activation and apoptosis. This thus suggested a potential dual role for PKC␦ in ROS-induced LMVEC apoptosis, both as an upstream regulator of p38 activation and as an inducer of DNA-damage, through its caspase-3-dependent cleavage fragment (Grinnell et al., 2012). In contrast, other studies have not found any activation of p38 pathways with exposure to hyperoxia in MLE cells (Zhang et al., 2003), human fetal lung fibroblasts (Lang et al., 2010), and murine macrophages (Petrache et al., 1999). Recent data have also excluded p38 activation in Ang-II receptor activation (Jiang et al., 2012) and in Egr-1 induction in hyperoxia (Jones and Agani, 2003).

3. Conclusions The present literature review shows the wide involvement of MAPK signaling pathways in many aspects of lung responses to hyperoxia, and in many different cell types (i.e., alveolar cells, endothelial cells, fibroblasts, myofibroblasts, macrophages, mast cells). In vivo and in vitro experimental approaches demonstrates that hyperoxia activates ERK, JNK and p38 in lung cells. Most studies show pro-survival actions for ERK activation, and pro-apoptotic effects for JNK and p38 induction. However, it must also be stressed that many aspects remain controversial and need to be further addressed. Most studies discussed in the present review were performed in cell cultures; further analysis will be needed to better understand the roles of MAPK signaling in the more complex in vivo models. Previous studies have shown that oxidative-stress-related conditions, such as hyperoxic lung injury, can be treated with growth factors and anti-oxidant enzymes, such as heme oxygenase-1 and superoxide dismutase (e.g., Wispé et al., 1992; Ahmed et al., 2003; Ryter and Choi, 2005). Many studies have reported intriguing findings for the possibility of the modulation of tissue responses to hyperoxia through exogenous factors that involve ERK, JNK and p38. These studies have suggested the possibility to develop future therapeutic approaches aimed at reducing hyperoxic toxicity also through modulation of the components of the MAPK signaling pathways. The role of the administration of high levels of oxygen in the pathogenesis of lung pathologies such as BPD is well known, but the effects of neonatal hyperoxic exposure on other organs have only been under investigation more recently. For instance, in animal models, hyperoxia has been shown to induce functional alterations in heart function (Bandali et al., 2004; Yzydorczyk et al., 2008; Velten et al., 2012). Increased production of ROS (Bandali et al., 2004) and changes in the expression and activity of various factors have also been reported, such as for phospholipase C (Nagasawa et al., 2003), PKC, VEGF, HIF-1␣, eNOS/iNOS (Zara et al., 2012), NF␬B, and Akt (Zara et al., 2013). Some of these factors (e.g., VEGF, HIF-1␣, eNOS, NF-␬B) (Marconi et al., 2014) and a series of liver enzymes (e.g., Okamoto et al., 1993; Miralles et al., 2000; Couroucli et al., 2002; Malleske et al., 2006) have been reported to change in response to hyperoxia. In the early postnatal period, supraphysiological concentrations of oxygen might affect the central nervous system (e.g., Hill et al., 2013), also with reference to neurogenic niches (Porzionato et al., 2013c) and the peripheral nervous system, mainly at the level of the carotid body (e.g., Di Giulio et al., 1998; De Caro et al., 2010, 2013; Dmitrieff et al., 2012; Porzionato et al., 2012b, 2013b). In these tissues, the role of the MAPK signaling pathways in responses to hyperoxia has not yet been fully examined. Considering the increasing findings on the MAPKs in the lung

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