2.2. CytosolicPhospholipase A ₂

There are three forms of PLA₂ in mammalian cells [24]. The first class of PLA₂ is secretary PLA₂ (sPLA₂) that is expressed in a variety of cell types [25] and it has no preference for AA at sn-2 position, requires millimolar amounts of Ca²⁺ for activity and is sensitive to sulfhydryl reducing agents, such as dithiothreitol (DTT), and is resistant to heat or acid conditions [25]. The second class of PLA₂ is calcium-independent PLA₂ (iPLA₂) that does not require Ca²⁺ for catalytic activity. iPLA₂ prefers plasmalogen substrates and does not appear to have a preference for the type of fatty acid at the sn-2 position [26]. The third class is the novel, high molecular weight (85kDa) cytosolic PLA₂ (cPLA₂). cPLA₂ enzymes catalyze the hydrolysis of the sn-2 position of membrane glycerophospholipids, leading to production of free fatty acids and lysophospholipids. This reaction is of particular importance if the esterified fatty acid is arachidonic acid (AA) [24], which is converted by downstream metabolic enzymes to various bioactive lipophilic compounds called eicosanoids, including prostaglandins (PGs) and leukotrienes (LTs) [24]. PLA₂ could be the initial and rate-limiting enzyme in this conversion. The increase in cPLA₂ activation and expression following external stimuli, including proinflammatory cytokines, growth factors, and microbial toxin, is often observed in several systems [27]. The implication of cPLA₂ in inflammatory diseases has been confirmed by that the airway anaphylactic response in the cPLA₂ knockout mice is markedly reduced compared with that in the wild-type mice [28]. Moreover, cPLA₂-deficient mice have provided the most definitive evidence for the central role of cPLA₂ in eicosanoid [29] as well as in the pathogenesis of several inflammatory diseases, such as acute respiratory distress syndrome (ARDS) due to bacterial sepsis [30, 31]. These studies have demonstrated that there was a reduction in the bronchial lumen and alveolar thickening in the control mice that was remarkably absent in the cPLA₂ knockout mice. This outcome also appeared in 5-lipoxygenase (LO)-knockout mice and mice with PGD₂ receptor deficiency [32]. Thus, cPLA₂ seems to function as a crucial upstream regulator of the production of eicosanoids for airway resistance during allergic inflammation and is correlated to the process of asthma (Figure 1). The inhibition of cPLA₂-mediated pathways may also provide a therapeutic approach to airway and lung injury.

2.3. Cyclooxygenase-2

COX metabolites have diverse effects in the lung and are known to modify airway tone as well as inflammatory responses [3]. Three isoforms of COX have been identified [24]. COX-1 is constitutively expressed in most tissues and considered to be the “housekeeping” isoform that produces PGs which are required for maintenance of normal cell and organ function. In contrast, COX-2 is primarily an inducible isoform whose expression can be upregulated in many cell types by cytokines, mitogens, and endotoxin [3, 4]. It is highly expressed in inflamed tissues and believed to produce PGs involved in inflammatory processes [33]. COX-2 has multiple transcriptional regulatory sequences in its promoter region, including a TATA box, an NF-IL6 motif, two AP-2 sites, three Sp1 sites, two NF-κB sites, a CRE motif, and an E-box [34]. COX-2 gene expression can be induced by multiple cytokines and growth factors, via activation of transcriptional regulatory proteins that act on these promoter sites [35]. Thus, COX-2 appears to be the primary COX controlling PGE₂ synthesis in response to inflammation (Figure 1). COX effects are widespread and extremely complex; however, studies in knockout mice for COX-1 versus COX-2 reveal sometimes overlapping, not altogether predictable roles for these two enzymes [24]. The levels of prostanoids in bronchoalveolar lavage fluid are increased in asthma, and several studies have found enhanced expression of both COX-1 and COX-2 in the airways of asthmatics [36, 37]. A recent research has renewed interest in the role of prostanoids in allergic airway disease. Moreover, the expression of COX-2 protein induced by Lactobacillus rhamnosus GG (LGG), endotoxin, and lipoteichoic acid (LTA) in T84 epithelial cells [38]. The presence of COX-3 mRNA transcript, with a size of approximately 5.2kb, was subsequently confirmed in human cells; COX-3 was in highest concentrations in the cerebral cortex and heart tissue [24]. The regulation of COX-3 transcription appears to be identical to that of COX-1. COX-3 is similar to COX-1 and COX-2 in terms of structure and enzymatic function. However, the retention of intron 1 in COX-3 seems to slow its enzymatic activity in comparison to COX-1 and COX-2. Thus, the inhibition of COX-2-mediated inflammatory pathway may provide a therapeutic approach to respiratory diseases.

3.2. NADPHOxidase/ROS

ROS are products of normal cellular metabolism and are known to act as second messengers. Under physiological conditions, ROS participate in maintenance of cellular “redox homeostasis” to protect cells against oxidative stress. In addition, regulation of redox state is important for cell activation, viability, proliferation, and organ function. However, overproduction of ROS, most frequently due to excessive stimulation of either reduced NADPH by proinflammatory cytokines or the mitochondrial electron transport chain and xanthine oxidase, results in oxidative stress. Oxidative stress is a deleterious process that leads to airway and lung damage and consequently to several respiratory inflammatory diseases/injuries [8]. ROS are intracellularly generated from several sources, including mitochondrial respiration, cytochrome P450, the NADPH oxidase system, and xanthine/xanthine oxidase [54]. However, the major ROS generating enzyme is NADPH oxidase, a membrane-bound multicomponent enzyme complex that is present in phagocytes as well as nonphagocytic cells [55]. ROS produced by NADPH oxidase have two major roles. First, superoxide produced by NADPH oxidase 2 is required for respiratory burst that occurs in phagocytes, leading to microbial killing. The second role of NADPH oxidase is associated with the regulation of cell signaling [55]. ROS derived from NADPH oxidase can specifically and reversibly react with proteins, altering their activity, localization, and half-life [8]. Activated phagocytic cells generate ROS via assembly and activation of the NADPH oxidase complex, which comprises membrane-associated flavocytochrome b₅₅₈ (gp91^phox) and p22^phox and various cytosolic cofactors (p47^phox, p67^phox, and p40^phox, and the GTPase, Rac1) and mediates transmembrane electron transfer from the major cellular electron donor, NADPH, to reduce molecular O₂ to superoxide anion (O₂ ^•−) and hydrogen peroxide (H₂O₂) [54]. A number of homologs of the main business end of NADPH oxidase, gp91^phox, have been discovered, and mammalian systems are now known to contain seven NADPH oxidase homologs, comprising NADPH oxidase 1–5 (NADPH oxidase 2 being the new name for gp91^phox) and two larger dual oxidases, DUOX1 and DUOX2, which are widely expressed in many cell types to mediate a variety of biological functions, such as cell mitosis, differentiation, migration, and immune regulation [56]. In in vitro studies, using macrophages, alveolar and bronchial epithelial cells, ROS have been shown to induce gene expression of inflammatory mediators, such as IL-1 and TNF-α [57, 58]. Patients with asthma demonstrate increased generation of ROS, such as superoxide anion, hydrogen peroxide, and hydroxyl radicals. Increased production of ROS has been demonstrated by many cell types within the lung in asthma, including macrophages, antigen-presenting cells (APCs), neutrophils, and eosinophils [59]. Excessive production of ROS correlates with the degree of airway hyperresponsiveness, as quantified by methacholine challenge. In addition, oxidative stress also contributes to a proteinase-antiproteinase imbalance, both by inactivating antiproteinases, such as α1-antitrypsin and secretory leukocyte proteinase inhibitor, and by activating proteinases, such as MMPs [8]. On the other hand, oxidants also promote inflammation by activating NF-κB or AP-1, which orchestrates the expression of multiple inflammatory genes recognized to be important in COPD, such as TNF-α. Recently, phagocytic NADPH oxidase-ROS signaling has been shown to play a critical role in promoting TNF-α-induced, NF-κB-dependent acute inflammatory responses, and tissue injury specifically in the lungs, which is effected by preferential leukocyte infiltration [8]. Thus, oxidative stress plays a critical role in inflammatory responses in airway and lung diseases via the upregulation of redox-sensitive transcription factors (such as AP-1 or NF-κB) and thereby proinflammatory genes (such as MMP-9, VCAM-1, ICAM-1, COX-2, or cPLA₂) expression. Inflammation itself results in oxidative stress in the airways and lungs. Taken together, NADPH oxidase/ROS play a critical role during development of airway and lung diseases (Figure 2).

3.3. PI3K/Akt

The PI3K family are central signaling elements in a diverse array of cellular functions, including growth, proliferation, migration, and survival. It is, therefore, understandable that dysregulation of PI3K has been implicated in the induction and/or progression of a variety of disease states, including those of the respiratory tract, ranging from asthma to cancer [60]. PI3Ks have been divided into three classes according to theirstructure and lipid substrate specificity [61]. The most extensively investigated are class I PI3Ks. Type I PI3Ks are activated by cell surface receptors, such as growth factors, insulin, and G-protein-coupled receptors (GPCRs). Class II PI3Ks comprised α, β, and γ isoforms, which are characterized by the presence of a C2 domain at the C terminus. They predominantly use phosphatidylinositol and phosphatidylinositol 4-phosphate [PI(4)P] as substrates. The class III PI3Ks only use phosphatidylinositol as a substrate. Class I PI3Ks are further divided into class IA and class IBPI3Ks. Structurally, PI3Ks IA exist as heterodimeric complexes in which a catalytic p110 subunit (designated as α, β, or γ) is in association with a particular regulatory subunit (designated as p85, p55, and p50) [61]. Importantly, PI3K IA signals downstream of receptor tyrosine kinase and Ras. The single class PI3K IB consists of the p110γ catalytic subunit complexed to the p101 regulatory subunit and signals downstream of GPCRs and Ras, which is activated by βγ subunits from GPCRs, such as the receptors for chemokines [61]. Despite limitations in selectivity, the two commercially available PI3K inhibitors, wortmannin and LY294002, have contributed greatly to our understanding of the biological role of PI3K in lung inflammation [62]. Moreover, previous study indicated that intratracheal administration of LY294002 significantly reduced ovalbumin-(OVA-) induced increases in total cell counts, eosinophil counts, and IL-5, IL-13, and CCL11 (eotaxin) levels in BAL fluid and dramatically inhibited OVA-induced tissue eosinophilia and airway mucus production [63]. This study confirmed that LY294002 markedly attenuated OVA-induced serine phosphorylation of Akt, a direct downstream substrate of PI3K. In addition, other studies also showed that LY294002 and wortmannin attenuated eosinophilic airway inflammation and airway hyperresponsiveness in a murine asthma model [64]. Thus, PI3K inhibition was indicated to have therapeutic potential for the treatment of asthmatic airway inflammation. On the other hand, ROS induction is accompanied by activation of PI3K. LY294002 was shown to reduce chemokine-induced ROS generation in phagocytes, which was further confirmed by studies using PI3K knockout mice [62]. It was also reported that serum withdrawal (SW) killed human U937 blood cells by elevating cellular ROS levels, which occurred through PI3K activation [65]. Thus, PI3K family plays a prominent role in various airway and lung inflammation (Figure 2). Moreover, inhibitors of PI3K/Akt may prove to be useful novel therapies in the treatment of respiratory diseases.

3.4. SrcFamilyKinases

SFKs are signaling enzymes that have long been recognized to regulate critical cellular processes, such as proliferation, survival, migration, and metastasis [8]. Src protein tyrosine kinase (PTK) family is categorized into nonreceptor tyrosine kinases and consists of nine members. Src, Fyn, Yes, and Yrk are ubiquitously expressed, whereas Blk, Fgr, Hck, Lck, and Lyn are expressed in more restricted patterns [66]. So far, Yrk has been detected only in chicken [66]. Src PTK family members are activated in response to the stimulation of a variety of cell surface receptors, such as tyrosine kinase receptors, integrin receptors, and G protein-coupledreceptors, and by cellular stress [66]. Src PTKs can also regulate the functional activity of these receptors. Moreover, we reported that TNF-α or IL-1β induces VCAM-1 and ICAM-1 expression via a c-Src-dependent pathway in human airway smooth muscle cells [8]. In addition, c-Src has been shown to regulate COX-2/PGE₂/IL-6-dependent airway inflammation via NADPH oxidase/ROS [67]. In human lung epithelial cells, in addition to activating NF-κB-inducing kinase (NIK) via TRAF2, TNF-α could activate c-Src through PKC. Systemic inhibition of these kinases using specific small molecule inhibitors for Src PTKs (either PP2 or SU-6656) significantly attenuated LPS-induced lung injury and capillary permeability and reduced LPS-dependent cytokine and chemokine levels in the lung and the serum [68]. Thus, the role of Src family PTKs in inflammatory responses is a rising area of research (Figure 2). However, application of small chemical inhibitors to effectively and specifically block Src PTKs could have a great clinical implication for airway and lung diseases with inflammatory responses as underlying mechanisms.

3.5. GrowthFactorTyrosineKinaseReceptors

Cell-surface tyrosine kinases receptors play pivotal roles in development, tissue repair, and normal cellular homeostasis. Aberrant expression or signaling patterns of these receptors have also been linked to the progression of a diversity of diseases, including asthma. Two major families of tyrosine kinases receptors, the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) families, have received a great deal of attention as potentially therapeutic targets for respiratory diseases, as these receptors have been shown to play key roles in chronic tissue remodeling in asthma, bronchitis, and pulmonary fibrosis. The EGFR system on epithelial cells and underlying mesenchymal cells (fibroblasts, myofibroblasts, and smooth muscle cells) drives numerous phenotypic changes during the progression of these pulmonary diseases, including mesenchymal cell hyperplasia, differentiation, and ECM production. The PDGFR system, located primarily on mesenchymal cells, transduces signals for cell survival, growth, and chemotaxis. The variety of EGFR and PDGFR ligands produced by the airway epithelium or adjacent mesenchymal cells allows for intimate epithelial-mesenchymal cell communication. In humans, the airway epithelium expresses EGFR ligands constitutively, including EGF, TGF-α, HB-EGF, amphiregulin, heregulin, and betacellulin. Expression of several EGFR ligands has also been investigated in diseases, such as COPD and asthma. Kohri et al. showed that P. aeruginosa bacterial supernatant induces mucin production in human airway epithelial cells (NCI-H292) via EGFR activation [69]. Multiple studies have also reported that stimulation of airway epithelial cells by LPS induces the secretion of IL-8 via a cellular cascade involving a TLR4/myeloid differentiation primary response gene (MyD)88/NF-κB-dependent pathway [70]. In addition, ROS have been shown to stimulate PDGFRα activation via c-Src family kinases [8]. There is accumulating evidence that PKC-dependent phosphorylation of p47^phox is essential for PDGF-stimulated ROS generation, which is important for PDGF-induced MAPKs activation [8]. Taken together, these studies suggest that growth factor tyrosine kinase receptors may also play a key role in mediating expression of inflammatory genes (Figure 2).

3.6. Mitogen-ActivatedProteinKinases

MAPKs are important components of signaling modules activated by neurotransmitters, cytokines, and growth factors, as well as chemical and mechanical stressors. In the airway, these external signals produce acute responses that modify smooth muscle contraction and may also induce chronic responses that modify airway structure [71]. Both acute and chronic events in airway remodeling result from altered expression of multiple genes encoding protein mediators of cell-cell signaling, ECM remodeling, cell cycle control, and intracellular signaling pathways [72]. In mammals, three groups of MAPKs have been identified: the extracellular signal-regulated protein kinases (ERKs), the c-Jun NH₂-terminal kinases (JNKs), and the p38 MAPK. ERK is activated by diverse stimuli, including growth factors and cytokines [73]. The p38 MAPK is activated by cellular stresses, including UV radiation, LPS, growth factors, and cytokines. The JNK is activated by many of the same stimuli that activate p38 MAPK, such as cellular stresses and numerous cytokines. Thus, the inhibition of MAPKs activity via pharmacological or genetic approaches blocks allergic inflammation of airways. Moreover, asthmatic patients demonstrated increased immunostaining for phospho (p)-ERK, p-p38 MAPK, and p-JNK [72]. p-ERK staining was observed especially in airway epithelium and smooth muscle cells. The phosphorylation of p38 MAPK was primarily observed in the basal layer of the columnar epithelium. It is likely that p38 MAPK drives basal metabolic processes for this particular cell type. There was significant correlation between clinical severity of asthma and intensity of immunostaining for p-ERK and p-p38 MAPK and between p-ERK and the number of tissue eosinophils and neutrophils in the airways. p-JNK primarily stained airway smooth muscle cells. Early studies of p38 MAPK demonstrated that IL-1β and TNF-α activate the p38 MAPK in monocytes [74]. Furthermore, inhibition of the p38 MAPK pathway was shown to exert anti-inflammatory effects through inhibition of IL-1β, IL-6, and TNF-α expression [75]. In airway smooth muscle cells, there is solid evidence that both ERK and p38 MAPK pathways contribute to IL-1β-induced COX-2 expression and PGE₂ synthesis [76, 77]. In a mouse model of chronic lung inflammation (allergic inflammation), significant inhibition of TNF-α, IL-4, IL-13, and RANTES (regulated on activation, normal T-cell expressed and secreted) in lung homogenates was observed with JNK inhibitor, SP600125 [78]. In addition, we also found that LTA or IL-1β could induce cPLA₂, COX-2, or MMP-9 in human airway smooth muscle cells or A549 cells [3, 44]. Therefore, MAPKs play an important role in mediating airway and lung inflammation (Figure 2).

3.7. NF-κB

NF-κB is viewed as a master regulator of inflammatory responses because it plays an essential role in the evolution as well as the resolution phase of inflammation. NF-κB controls a wide spectrum of biological effects ranging from immune and stress-induced responses to cell fate decisions such as proliferation, differentiation, tumorigenesis, apoptosis, and tissue remodeling [8]. NF-κB usually exists as a heterodimeric complex of p50 and p65/RelA subunits. In unstimulated cells, NF-κB is found in the cytoplasm as an inactive non-DNA-binding form, associated with an inhibitor protein called inhibitory κB (IκB) which masks the nuclear translocation signal and so prevents NF-κB from entering the nucleus. Upon cell stimulation with various NF-κB inducers, IκBα is rapidly phosphorylated on two serine residues, which targets the inhibitor protein for ubiquitination by the E3 ubiquitin-ligases (E3RSIκB) and subsequent degradation by the 26S proteasome [79]. The released NF-κB dimer can then be translocated into the nucleus and activate target genes by binding with high affinity to κB elements in their promoters. NF-κB is activated by numerous extracellular stimuli, including cytokines such as TNF-α and IL-1β, viruses and environmental particulates (PM10s), and oxidative stress [8]. Exogenous H₂O₂ also activated NF-κB in a murine model of ROS-induced acute lung injury. Administration of OTC (L-2-oxothiazolidine-4-carboxylate) resulted in significant reduction of NF-κB translocation into the nucleus and expression of adhesion molecules, chemokines, and cytokines [80]. Previous study demonstrated that NF-κB activation occurred rapidly in the ovalbumin (OVA) model of allergic airways disease and that NF-κB activation predominantly occurred in the epithelial cells of the conducting airways, in association with enhanced mRNA expression of NF-κB-regulated chemokine genes, including MIP-2 and eotaxin [81]. A novel cyclin-dependent kinase inhibitor (BAI) has been shown to downregulate TNF-α-induced expression of cell adhesion molecules by inhibition of NF-κB activation in human pulmonary epithelial cells [82]. Recently, we also demonstrated that overexpression of HO-1 protects against TNF-α-mediated airway inflammation by downregulation of TNFR1-dependent oxidative stress and NF-κB activation [83]. Taken together, these results show that NF-κB plays a key role in mediating the expression of inflammatory proteins in airway and lung inflammation and injury (Figure 2).

The authors declare that they have no conflict of interests.