Collagen deposition is observed in a diverse set of pulmonary diseases, and the unraveling of the molecular signaling pathways that facilitate collagen deposition represents an ongoing area of investigation. The stress-activated protein kinase, c-Jun N-terminal kinase 1 (JNK1), is activated by a large variety of cellular stresses and environmental insults. Recent work from our laboratory demonstrated the critical role of JNK1 in epithelial to mesenchymal transition. The goal of the present study was to examine the involvement of JNK1 in subepithelial collagen deposition in mice subjected to models of allergic airways disease and interstitial pulmonary fibrosis. Activation of JNK was slightly enhanced in lungs from mice subjected to sensitization and challenge with ovalbumin (Ova), and predominant localization of phospho-JNK was observed in the bronchial epithelium. While mice lacking JNK1 (JNK1−/− mice) displayed enhanced lung inflammation and cytokine production compared with wild-type (WT) mice, JNK1−/− mice accumulated less subepithelial collagen deposition in response to antigen, and showed decreased expression of profibrotic genes compared with WT animals. Furthermore, transforming growth factor (TGF)-β1 content in the bronchoalveolar lavage was diminished in JNK1−/− mice compared with WT animals subjected to antigen. Finally, we demonstrated that mice lacking JNK1 were protected against TGF-β1 and bleomycin-induced pro-fibrotic gene expression and pulmonary fibrosis. Collectively, these findings demonstrate an important requirement for JNK1 in promoting collagen deposition in multiple models of fibrosis.
TGF-β; ovalbumin; asthma; fibrosis; bleomycin
Oxidative stress is a hallmark of asthma, and increased levels of oxidants are considered markers of the inflammatory process. Most studies to date addressing the role of oxidants in the etiology of asthma were based on the therapeutic administration of low m.w. antioxidants or antioxidant mimetic compounds. To directly address the function of endogenous hydrogen peroxide in the pathophysiology of allergic airway disease, we comparatively evaluated mice systemically overexpressing catalase, a major antioxidant enzyme that detoxifies hydrogen peroxide, and C57BL/6 strain matched controls in the OVA model of allergic airways disease. Catalase transgenic mice had 8-fold increases in catalase activity in lung tissue, and had lowered DCF oxidation in tracheal epithelial cells, compared with C57BL/6 controls. Despite these differences, both strains showed similar increases in OVA-specific IgE, IgG1, and IgG2a levels, comparable airway and tissue inflammation, and identical increases in procollagen 1 mRNA expression, following sensitization and challenge with OVA. Unexpectedly, mRNA expression of MUC5AC and CLCA3 genes were enhanced in catalase transgenic mice, compared with C57BL/6 mice subjected to Ag. Furthermore, when compared with control mice, catalase overexpression increased airway hyperresponsiveness to methacholine both in naive mice as well as in response to Ag. In contrast to the prevailing notion that hydrogen peroxide is positively associated with the etiology of allergic airways disease, the current findings suggest that endogenous hydrogen peroxide serves a role in suppressing both mucus production and airway hyperresponsiveness.
Glutaredoxins (GRX) are antioxidant enzymes that preferentially catalyze the reduction of protein-glutathione mixed disulfides. The formation of mixed disulfides with GSH is known as S-glutathionylation, a post-translational modification that is emerging as an important mode of redox signaling. Since asthma is a disease that is associated with increased oxidative stress and altered antioxidant defenses, we investigated the expression of GRX in a murine model of allergic airway disease. Sensitization and challenge of C57BL/6 mice with ovalbumin resulted in increased expression of GRX1 mRNA, as well as increased amounts of GRX1 protein and total GRX activity in the lung. Because GRX1 expression is prominent in bronchial epithelium, we isolated primary epithelial cells from mouse trachea to investigate the presence of GRX. Primary tracheal epithelial cells were found to express both GRX1 and 2 mRNA and detectable GRX activity. Treatment with IFN-γ increased the expression of GRX1 and overall GRX activity, resulting in attenuation of protein S-glutathionylation. In contrast, TGF-β1 caused decreased GRX1 expression and overall GRX activity, leading to markedly enhanced protein S-glutathionylation. GRX1 joins the cadre of antioxidant defenses known to be modulated during allergic airway inflammation.
glutaredoxin; asthma; epithelium; IFN-γ; TGF-β
The transcription factor NF-κB has been causally linked to inflammatory lung diseases. Recent studies have unraveled the complexity of NF-κB activation by identifying two parallel activation pathways: the classical NF-κB pathway, which is controlled by IκB kinase complex–β (IKKβ) and RelA/p50, and the alternative pathway, which is controlled by IKKα and RelB/p52. The alternative pathway regulates adaptive immune responses and lymphoid development, yet its role in the regulation of innate immune responses remains largely unknown. In this study, we determined the relevance of the alternative NF-κB pathway in proinflammatory responses in lung epithelial cells. The exposure of C10 murine alveolar lung epithelial cells to diverse stimuli, or primary murine tracheal epithelial cells to LPS, resulted in the activation of both NF-κB pathways, based on the nuclear translocation of RelA, p50, RelB, and p52. Increases in the nuclear content of RelA occurred rapidly, but transiently, whereas increases in nuclear RelB content were protracted. The small interfering (si) RNA–mediated knockdown of IKKα, RelA, or RelB resulted in decreases of multiple LPS-induced proinflammatory cytokines. Surprisingly, the siRNA ablation of IKKα or RelB led to marked increases in the production of IL-6 in response to LPS. The simultaneous expression of constitutively active (CA)-IKKα and CA-IKKβ caused synergistic increases in proinflammatory mediators. Lastly, the disruption of the IKK signalsome inhibited the activation of both NF-κB pathways. These results demonstrate that the coordinated activation of both NF-κB pathways regulates the magnitude and nature of proinflammatory responses in lung epithelial cells.
lung; IκB kinase–β; IκB kinase–α; RelA; RelB
Recent studies suggest the importance of the transition of airway epithelial cells (EMT) in pulmonary fibrosis, and also indicate a role for Wingless protein (Wnt)/β-catenin signaling in idiopathic pulmonary fibrosis. We investigated the possible role of the Wnt signaling pathway in inducing EMT in lung epithelial cells, and sought to unravel the role of c-Jun–N-terminal-kinase–1 (JNK1). The exposure of C10 lung epithelial cells or primary mouse tracheal epithelial cells (MTECs) to Wnt3a resulted in increases in JNK phosphorylation and nuclear β-catenin content. Because the role of β-catenin as a transcriptional coactivator is well established, we investigated T-cell factor/lymphocyte-enhancement factor (TCF/LEF) transcriptional activity in C10 lung epithelial cells after the activation of Wnt. TCF/LEF transcriptional activity was enhanced after the activation of Wnt, and this increase in TCF/LEF transcriptional activity was diminished after the small interfering (si)RNA-mediated ablation of JNK. The activation of the Wnt pathway by Wnt3a, or the expression of either wild-type or constitutively active β-catenin (S37A), led to the activation of an EMT transcriptome, manifested by the increased mRNA expression of CArG box-binding factor–A, fibroblast-specific protein (FSP)–1, α–smooth muscle actin (α-SMA), and vimentin, increases in the content of α-SMA and FSP1, and the concomitant loss of zona occludens–1. The siRNA-mediated ablation of β-catenin substantially decreased Wnt3a-induced EMT. The siRNA ablation of JNK1 largely abolished Wnt3a, β-catenin, and β-catenin S37a-induced EMT. In MTECs lacking Jnk1, Wnt3a-induced increases in nuclear β-catenin, EMT transcriptome, and the content of α-SMA or FSP1 were substantially diminished. These data show that the activation of the Wnt signaling pathway is capable of inducing an EMT program in lung epithelial cells through β-catenin, and that this process is controlled by JNK1.
lung; epithelium; Wnt3a; fibrosis; epithelial to mesenchymal transition
Significance: Redox-based signaling governs a number of important pathways in tissue homeostasis. Consequently, deregulation of redox-controlled processes has been linked to a number of human diseases. Among the biological processes regulated by redox signaling, apoptosis or programmed cell death is a highly conserved process important for tissue homeostasis. Apoptosis can be triggered by a wide variety of stimuli, including death receptor ligands, environmental agents, and cytotoxic drugs. Apoptosis has also been implicated in the etiology of many human diseases. Recent Advances: Recent discoveries demonstrate that redox-based changes are required for efficient activation of apoptosis. Among these redox changes, alterations in the abundant thiol, glutathione (GSH), and the oxidative post-translational modification, protein S-glutathionylation (PSSG) have come to the forefront as critical regulators of apoptosis. Critical Issues: Although redox-based changes have been documented in apoptosis and disease pathogenesis, the mechanistic details, whereby redox perturbations intersect with pathogenic processes, remain obscure. Future Directions: Further research will be needed to understand the context in which of the members of the death receptor pathways undergo ligand dependent oxidative modifications. Additional investigation into the interplay between oxidative modifications, redox enzymes, and apoptosis pathway members are also critically needed to improve our understanding how redox-based control is achieved. Such analyses will be important in understanding the diverse chronic diseases. In this review we will discuss the emerging paradigms in our current understanding of redox-based regulation of apoptosis with an emphasis on S-glutathionylation of proteins and the enzymes involved in this important post-translational modification. Antioxid. Redox Signal. 16, 496–505.
We recently demonstrated that S-glutathionylation of the death receptor Fas (Fas-SSG) amplifies apoptosis (V. Anathy et al., J. Cell Biol. 184:241–252, 2009). In the present study, we demonstrate that distinct pools of Fas exist in cells. Upon ligation of surface Fas, a separate pool of latent Fas in the endoplasmic reticulum (ER) underwent rapid oxidative processing characterized by the loss of free sulfhydryl content (Fas-SH) and resultant increases in S-glutathionylation of Cys294, leading to increases of surface Fas. Stimulation with FasL rapidly induced associations of Fas with ERp57 and glutathione S-transferase π (GSTP), a protein disulfide isomerase and catalyst of S-glutathionylation, respectively, in the ER. Knockdown or inhibition of ERp57 and GSTP1 substantially decreased FasL-induced oxidative processing and S-glutathionylation of Fas, resulting in decreased death-inducing signaling complex formation and caspase activity and enhanced survival. Bleomycin-induced pulmonary fibrosis was accompanied by increased interactions between Fas-ERp57-GSTP1 and S-glutathionylation of Fas. Importantly, fibrosis was largely prevented following short interfering RNA-mediated ablation of ERp57 and GSTP. Collectively, these findings illuminate a regulatory switch, a ligand-initiated oxidative processing of latent Fas, that controls the strength of apoptosis.
It is established that cigarette smoke (CS) causes irreversible oxidations in lung epithelial cells, and can lead to their death. However, its impact on reversible and physiologically relevant redox-dependent protein modifications remains to be investigated. Glutathione is an important antioxidant against inhaled reactive oxygen species as a direct scavenger, but it can also covalently bind protein thiols upon mild oxidative stress to protect them against irreversible oxidation. This posttranslational modification, known as S-glutathionylation, can be reversed under physiological conditions by the enzyme, glutaredoxin 1 (Grx1). The aim of this study was to investigate if CS modifies Grx1, and if this impacts on protein S-glutathionylation and epithelial cell death. Upon exposure of alveolar epithelial cells to CS extract (CSE), a decrease in Grx1 mRNA and protein expression was observed, in conjunction with decreased activity and increased protein S-glutathionylation. Using mass spectrometry, irreversible oxidation of recombinant Grx1 by CSE and acrolein was demonstrated, which was associated with attenuated enzyme activity. Furthermore, carbonylation of Grx1 in epithelial cells after exposure to CSE was shown. Overexpression of Grx1 attenuated CSE-induced increases in protein S-glutathionylation and increased survival. Conversely, primary tracheal epithelial cells of mice lacking Grx1 were more sensitive to CS-induced cell death, with corresponding increases in protein S-glutathionylation. These results show that CS can modulate Grx1, not only at the expression level, but can also directly modify Grx1 itself, decreasing its activity. These findings demonstrate a role for the Grx1/S-glutathionylation redox system in CS-induced lung epithelial cell death.
chronic obstructive pulmonary disease; cigarette smoke; cell death; glutaredoxin; protein S-glutathionylation
The transcription factor, Nuclear Factor kappa B (NF-κB) is a critical regulator of inflammation and immunity, and is negatively regulated via S-glutathionylation. The inhibitory effect of S-glutathionylation is overcome by glutaredoxin-1 (Grx1), which under physiological conditions catalyses deglutathionylation and enhances NF-κB activation. The mechanisms whereby expression of the Glrx1 gene is regulated remain unknown. Here we examined the role of NF-κB in regulating activation of Glrx1. Transgenic mice which express a doxycyclin-inducible constitutively active version of inhibitory kappa B kinase-beta (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2kb region Glrx1 promoter that contains two putative NF-κB binding sites was activated by CA-IKKβ, RelA/p50, and lipopolysaccharide (LPS). Chromatin immunoprecipitation experiments confirmed binding of RelA to the promoter of Glrx1 in response to LPS. Stimulation of C10 lung epithelial cells with LPS caused transient increases in Grx1 mRNA expression, and time-dependent increases in S-glutathionylation of IKKβ. Overexpression of Grx1 decreased S-glutathionylation of IKKβ, prolonged NF-κB activation, and increased levels of pro-inflammatory mediators. Collectively, this study demonstrates that the Glrx1 gene is positively regulated by NF-κB, and suggests a feed forward mechanism to promote NF-κB signaling by decreasing S-glutathionylation.
S-glutathionylation; Nuclear Factor kappa B; Glutaredoxin; Lung; Inhibitory kappa B kinase
Activation of NF-κB in airway epithelium is observed in allergic asthma and is induced by inhalation of numerous infectious and reactive substances. Many of the substances that activate NF-κB in the airway epithelium are also capable of acting as adjuvants to elicit antigen-specific sensitization to concomitantly inhaled protein, thereby circumventing the inherent bias of the lung to promote tolerance to innocuous antigens. We have used a transgenic mouse inducibly expressing a constitutively active mutant of the inhibitor of nuclear factor κB (IκB) kinase β (CAIKKβ) that activates NF-κB only in nonciliated airway epithelial cells to test whether activation of this intracellular signaling pathway in this specific cell type is sufficient to establish a pulmonary environment permissive to the development of allergic sensitization to inhaled protein. When airway epithelial CAIKKβ was transiently expressed in antigen-naive mice only during initial inhalation of ovalbumin, the mice became allergically sensitized to the antigen. As a consequence, subsequent inhalation of ovalbumin alone led to an allergic asthma–like response that included airway hyperresponsiveness to methacholine, eosinophilia, mucus expression, elevated serum levels of antigen-specific IgE and IgG1, and splenic CD4+ T cells that secreted T helper type 2 and type 17 cytokines in response to in vitro antigen restimulation. Furthermore, CD11c+ cells in the mediastinal lymph nodes (MLN) of CAIKKβ-expressing mice displayed significantly elevated levels of activation markers. These data implicate airway epithelial NF-κB activation as a critical modulator of the adaptive immune response to inhaled antigens via the secretion of soluble mediators that affect the capacity of CD11c+ cells to undergo maturation and promote antigen-specific allergic responses.
epithelial cell; antigen-presenting cell; NF-κB; allergy; asthma
Protein S-glutathionylation (PSSG), a reversible posttranslational modification of reactive cysteines, recently emerged as a regulatory mechanism that affects diverse cell-signaling cascades. The extent of cellular PSSG is controlled by the oxidoreductase glutaredoxin-1 (Grx1), a cytosolic enzyme that specifically de-glutathionylates proteins. Here, we sought to evaluate the impact of the genetic ablation of Grx1 on PSSG and on LPS-induced lung inflammation. In response to LPS, Grx1 activity increased in lung tissue and bronchoalveolar lavage (BAL) fluid in WT (WT) mice compared with PBS control mice. Glrx1−/− mice consistently showed slight but statistically insignificant decreases in total numbers of inflammatory cells recovered by BAL. However, LPS-induced concentrations of IL-1β, TNF-α, IL-6, and Granulocyte/Monocyte Colony–Stimulating Factor (GM-CSF) in BAL were significantly decreased in Glrx1−/− mice compared with WT mice. An in situ assessment of PSSG reactivity and a biochemical evaluation of PSSG content demonstrated increases in the lung tissue of Glrx1−/− animals in response to LPS, compared with WT mice or PBS control mice. We also demonstrated that PSSG reactivity was prominent in alveolar macrophages (AMs). Comparative BAL analyses from WT and Glrx1−/− mice revealed fewer and smaller AMs in Glrx1−/− mice, which showed a significantly decreased expression of NF-κB family members, impaired nuclear translocation of RelA, and lower levels of NF-κB–dependent cytokines after exposure to LPS, compared with WT cells. Taken together, these results indicate that Grx1 regulates the production of inflammatory mediators through control of S-glutathionylation–sensitive signaling pathways such as NF-κB, and that Grx1 expression is critical to the activation of AMs.
lipopolysaccharide; glutaredoxin-1; protein S-glutathionylation; nuclear factor-κB
Transforming growth factor (TGF)–β1 is a key mediator of lung remodeling and fibrosis. Epithelial cells are both a source of and can respond to TGF-β1 with epithelial-to-mesenchymal transition (EMT). We recently determined that TGF-β1–induced EMT in lung epithelial cells requires the presence of c-Jun N-terminal kinase (JNK) 1. Because TGF-β1 signals via Smad complexes, the goal of the present study was to determine the impact of JNK1 on phosphorylation of Smad3 and Smad3-dependent transcriptional responses in lung epithelial cells. Evaluation of JNK1-deficient lung epithelial cells demonstrated that TGF-β1–induced terminal phosphorylation of Smad3 was similar, whereas phosphorylation of mitogen-activated protein kinase sites in the linker regions of Smad3 was diminished, in JNK1-deficient cells compared with wild-type cells. In comparison to wild-type Smad3, expression of a mutant Smad3 in which linker mitogen-activated protein kinase sites were ablated caused a marked attenuation in JNK1 or TGF-β1–induced Smad-binding element transcriptional activity, and expression of plasminogen activator inhibitor–1, fibronectin-1, high-mobility group A2, CArG box–binding factor–A, and fibroblast-specific protein–1, genes critical in the process of EMT. JNK1 enhanced the interaction between Smad3 and Smad4, which depended on linker phosphorylation of Smad3. Conversely, Smad3 with phosphomimetic mutations in the linker domain further enhanced EMT-related genes and proteins, even in the absence of JNK1. Finally, we demonstrated a TGF-β1–induced interaction between Smad3 and JNK1. Collectively, these results demonstrate that Smad3 phosphorylation in the linker region and Smad transcriptional activity are directly or indirectly controlled by JNK1, and provide a putative mechanism whereby JNK1 promotes TGF-β1–induced EMT.
transforming growth factor–β1; c-Jun N-terminal kinase 1; Smad3; epithelial-to-mesenchymal transition
Reactive oxidants such as nitrogen dioxide (NO2) injure the pulmonary epithelium, causing airway damage and inflammation. We previously demonstrated that nuclear factor-κ B (NF-κB) activation within airway epithelial cells occurs in response to NO2 inhalation, and is critical for lipopolysaccharide-induced or antigen-induced inflammatory responses. Here, we investigated whether manipulation of NF-κB activity in lung epithelium affected severe lung injuries induced by NO2 inhalation. Wild-type C57BL/6J, CC10-IκBαSR transgenic mice with repressed airway epithelial NF-κB function, or transgenic mice expressing a doxycycline-inducible, constitutively active I κ B kinase β (CC10-rTet-CAIKKβ) with augmented NF-κB function in airway epithelium, were exposed to toxic levels of 25 ppm or 50 ppm NO2 for 6 hours a day for 1 or 3 days. In wild-type mice, NO2 caused the activation of NF-κB in airway epithelium after 6 hours, and after 3 days resulted in severe acute lung injury, characterized by neutrophilia, peribronchiolar lesions, and increased protein, lactate dehydrogenase, and inflammatory cytokines. Compared with wild-type mice, neutrophilic inflammation and elastase activity, lung injury, and several proinflammatory cytokines were significantly suppressed in CC10-IκBαSR mice exposed to 25 or 50 ppm NO2. Paradoxically, CC10-rTet-CAIKKβ mice that received doxycycline showed no further increase in NO2-induced lung injury compared with wild-type mice exposed to NO2, instead displaying significant reductions in histologic parameters of lung injury, despite elevations in several proinflammatory cytokines. These intriguing findings demonstrate distinct functions of airway epithelial NF-κB activities in oxidant-induced severe acute lung injury, and suggest that although airway epithelial NF-κB activities modulate NO2-induced pulmonary inflammation, additional NF-κB–regulated functions confer partial protection from lung injury.
epithelium; NF-κB; inflammation; nitrogen dioxide; lung injury
The oxidation of protein cysteine residues represents significant posttranslational modifications that impact a wide variety of signal transduction cascades and diverse biological processes. Oxidation of cysteines occurs through reactions with reactive oxygen as well as nitrogen species. These oxidative events can lead to irreversible modifications, such as the formation of sulfonic acids, or manifest as reversible modifications such as the conjugation of glutathione with the cysteine moiety, a process termed S-glutathionylation (also referred to as S-glutathiolation, or protein mixed disulfides). Similarly, S-nitrosothiols can also react with the thiol group in a process known as S-nitrosylation (or S-nitrosation). It is the latter two events that have recently come to the forefront of cellular biology through their ability to reversibly impact numerous cellular processes. Herein we describe two protocols for the detection of S-glutathionylated or S-nitrosylated proteins in situ. The protocol for the detection of S-glutathionylated proteins relies on the catalytic specificity of glutaredoxin-1 for the reduction of S-glutathionylated proteins. The protocol for the detection of S-nitrosylated proteins represents a modification of the previously described biotin switch protocol, which relies on ascorbate in the presence of chelators to decompose S-nitrosylated proteins. These techniques can be applied in situ to elucidate which compartments in tissues are affected in diseased states whose underlying pathologies are thought to represent a redox imbalance.
Fifteen years have passed since we published findings in the AJRCMB demonstrating that induction of early response fos/jun proto-oncogenes in rodent tracheal and mesothelial cells correlates with fibrous geometry and pathogenicity of asbestos. Our study was the first to suggest that the aberrant induction of signaling responses by crocidolite asbestos and erionite, a fibrous zeolite mineral associated with the development of malignant mesotheliomas (MMs) in areas of Turkey, led to altered gene expression. New data questioned the widely held belief at that time that the carcinogenic effects of asbestos in the development of lung cancer and MM were due to genotoxic or mutagenic effects. Later studies by our group revealed that proto-oncogene expression and several of the signaling pathways activated by asbestos were redox dependent, explaining why antioxidants and antioxidant enzymes were elevated in lung and pleura after exposure to asbestos and how they alleviated many of the phenotypic and functional effects of asbestos in vitro or after inhalation. Since these original studies, our efforts have expanded to understand the interface between asbestos-induced redox-dependent signal transduction cascades, the relationship between these pathways and cell fate, and the role of asbestos and cell interactions in development of asbestos-associated diseases. Of considerable significance is the fact that the signal transduction pathways activated by asbestos are also important in survival and chemoresistance of MMs and lung cancers. An understanding of the pathogenic features of asbestos fibers and dysregulation of signaling pathways allows strategies for the prevention and therapy of asbestos-related diseases.
proto-oncogenes; mitogen-activated protein kinases; epidermal growth factor receptor; activator protein-1; nuclear factor-κB
Tissue fibrosis is believed to be a manifestation of dysregulated repair following injury, in association with impaired reepithelialization, and aberrant myofibroblast activation and proliferation. Numerous pathways have been linked to the pathogenesis of fibrotic lung disease, including the death receptor Fas, which contributes to apoptosis of lung epithelial cells. A redox imbalance also has been implicated in disease pathogenesis, although mechanistic details whereby oxidative changes intersect with profibrotic signaling pathways remain elusive. Oxidation of cysteines in proteins, such as S-glutathionylation (PSSG), is known to act as a regulatory event that affects protein function. This manuscript will discuss evidence that S-glutathionylation regulates death receptor induced apoptosis, and the potential implications for cysteine oxidations in the pathogenesis of in fibrotic lung disease.
S-glutathionylation; glutaredoxin; Fas; apoptosis; fibrosis; lung
Pulmonary inflammation in asthma is orchestrated by the activity of NF-κB. NO and NO synthase (NOS) activity are important modulators of inflammation. The availability of the NOS substrate, l-arginine, is one of the mechanisms that controls the activity of NOS. Arginase also uses l-arginine as its substrate, and arginase-1 expression is highly induced in a murine model of asthma. Because we have previously described that arginase affects NOx content and interferes with the activation of NF-κB in lung epithelial cells, the goal of this study was to investigate the impact of arginase inhibition on the bioavailability of NO and the implications for NF-κB activation and inflammation in a mouse model of allergic airway disease. Administration of the arginase inhibitor BEC (S-(2-boronoethyl)-l-cysteine) decreased arginase activity and caused alterations in NO homeostasis, which were reflected by increases in S-nitrosylated and nitrated proteins in the lungs from inflamed mice. In contrast to our expectations, BEC enhanced perivascular and peribronchiolar lung inflammation, mucus metaplasia, NF-κB DNA binding, and mRNA expression of the NF-κB-driven chemokine genes CCL20 and KC, and lead to further increases in airways hyperresponsiveness. These results suggest that inhibition of arginase activity enhanced a variety of parameters relevant to allergic airways disease, possibly by altering NO homeostasis.
Transforming growth factor β1 (TGF-β1) is a cardinal cytokine in the pathogenesis of airway remodeling, and promotes epithelial-to-mesenchymal transition (EMT). As a molecular interaction between TGF-β1 and Jun N-terminal kinase (JNK) has been demonstrated, the goal of this study was to elucidate whether JNK plays a role in TGF-β1-induced EMT. Primary cultures of mouse tracheal epithelial cells (MTEC) from wild-type, JNK1–/– or JNK2–/– mice were comparatively evaluated for their ability to undergo EMT in response to TGF-β1. Wild-type MTEC exposed to TGF-β1 demonstrated a prominent induction of mesenchymal mediators and a loss of epithelial markers, in conjunction with a loss of trans-epithelial resistance (TER). Significantly, TGF-β1-mediated EMT was markedly blunted in epithelial cells lacking JNK1, while JNK2–/– MTEC underwent EMT in response to TGF-β1 in a similar way to wild-type cells. Although Smad2/3 phosphorylation and nuclear localization of Smad4 were similar in JNK1–/– MTEC in response to TGF-β1, Smad DNA-binding activity was diminished. Gene expression profiling demonstrated a global suppression of TGF-β1-modulated genes, including regulators of EMT in JNK1–/– MTEC, in comparison with wild-type cells. In aggregate, these results illuminate the novel role of airway epithelial-dependent JNK1 activation in EMT.
Lung; EMT; TGF-β; JNK; SMAD; Mouse
A wealth of recent studies points to the importance of airway epithelial cells in the orchestration of inflammatory responses in the allergic inflamed lung. Studies also point to a role of oxidative stress in the pathophysiology of chronic inflammatory diseases. This article provides a perspective on the significance of airway epithelial cells in allergic inflammation, and reviews the relevance of the transcription factor, nuclear factor κB, herein. We also provide the reader with a perspective on the role that oxidants can play in lung homeostasis, and address the concept of “redox biology.” In addition, we review recent evidence that highlights potential inhibitory roles of oxidants on nuclear factor κB activation and inflammation, and discuss recent assays that have become available to probe the functional roles of oxidants in lung biology.
epithelium; nuclear factor κB; asthma; oxidants
Airways display robust NF-κB activation and represent targets for anti-inflammatory asthma therapies, but the functional importance of NF-κB activation in airway epithelium remains enigmatic. Therefore, transgenic mice were created in which NF-κB activation is repressed specifically in airways (CC10-IκBαsr mice). In response to inhaled Ag, transgenic mice demonstrated significantly ameliorated inflammation, reduced levels of chemokines, T cell cytokines, mucus cell metaplasia, and circulating IgE compared with littermate controls. Despite these findings, Ag-driven airways hyperresponsiveness was not attenuated in CC10-IκBαsr mice. This study clearly demonstrates that airway epithelial NF-κB activation orchestrates Ag-induced inflammation and subsequent adaptive immune responses, but does not contribute to airways hyperresponsiveness, the cardinal feature that underlies asthma.
Reactive oxygen species (ROS) increase ligation of Fas (CD95), a receptor important for regulation of programmed cell death. Glutathionylation of reactive cysteines represents an oxidative modification that can be reversed by glutaredoxins (Grxs). The goal of this study was to determine whether Fas is redox regulated under physiological conditions. In this study, we demonstrate that stimulation with Fas ligand (FasL) induces S-glutathionylation of Fas at cysteine 294 independently of nicotinamide adenine dinucleotide phosphate reduced oxidase–induced ROS. Instead, Fas is S-glutathionylated after caspase-dependent degradation of Grx1, increasing subsequent caspase activation and apoptosis. Conversely, overexpression of Grx1 attenuates S-glutathionylation of Fas and partially protects against FasL-induced apoptosis. Redox-mediated Fas modification promotes its aggregation and recruitment into lipid rafts and enhances binding of FasL. As a result, death-inducing signaling complex formation is also increased, and subsequent activation of caspase-8 and -3 is augmented. These results define a novel redox-based mechanism to propagate Fas-dependent apoptosis.
oxidant; signal transduction; cysteine; S-glutathionylation; sulfenic acid; S-nitrosylation; peroxiredoxin; glutaredoxin; thioredoxin; biotin switch
Rationale: Nuclear factor (NF)-κB is a prominent proinflammatory transcription factor that plays a critical role in allergic airway disease. Previous studies demonstrated that inhibition of NF-κB in airway epithelium causes attenuation of allergic inflammation.
Objectives: We sought to determine if selective activation of NF-κB within the airway epithelium in the absence of other agonists is sufficient to cause allergic airway disease.
Methods: A transgenic mouse expressing a doxycycline (Dox)-inducible, constitutively active (CA) version of inhibitor of κB (IκB) kinase-β (IKKβ) under transcriptional control of the rat CC10 promoter, was generated.
Measurements and Main Results: After administration of Dox, expression of the CA-IKKβ transgene induced the nuclear translocation of RelA in airway epithelium. IKKβ-triggered activation of NF-κB led to an increased content of neutrophils and lymphocytes, and concomitant production of proinflammatory mediators, responses that were not observed in transgenic mice not receiving Dox, or in transgene-negative littermate control animals fed Dox. Unexpectedly, expression of the IKKβ transgene in airway epithelium was sufficient to cause airway hyperresponsiveness and smooth muscle thickening in absence of an antigen sensitization and challenge regimen, the presence of eosinophils, or the induction of mucus metaplasia.
Conclusions: These findings demonstrate that selective activation NF-κB in airway epithelium is sufficient to induce airway hyperresponsiveness and smooth muscle thickening, which are both critical features of allergic airway disease.
airway epithelium; nuclear factor-κB; inhibitory κB kinase-β; airway hyperresponsiveness; smooth muscle cell
Rationale: Asthma is characterized by increases in airway resistance, pulmonary remodeling, and lung inflammation. The cytokine transforming growth factor (TGF)-β has been shown to have a central role in asthma pathogenesis and in mouse models of allergic airway disease.
Objectives: To determine the contribution of TGF-β to airway hyperresponsiveness (AHR), we examined the time course, source, and isoform specificity of TGF-β production in an in vivo mouse asthma model. To then elucidate the function of TGF-β in AHR, inflammation, and pulmonary fibrosis, we examined the effects of blocking TGF-β signaling with neutralizing antibody.
Methods: Mice were sensitized and challenged with ovalbumin (OVA) to establish allergic airway disease. TGF-β activity was neutralized by intranasal administration of monoclonal antibody.
Measurements and Main Results: TGF-β1 protein levels were increased in OVA-challenged lungs versus naive controls, and airway epithelial cells were shown to be a likely source of TGF-β1. In addition, TGF-β1 levels were elevated in OVA-exposed IL-5–null mice, which fail to recruit eosinophils into the airways. Neutralization of TGF-β1 with specific antibody had no significant effect on airway inflammation and eosinophilia, although anti–TGF-β1 antibody enhanced OVA-induced AHR and suppressed pulmonary fibrosis.
Conclusions: These data show that TGF-β1 is the main TGF-β isoform produced after OVA challenge, with a likely cellular source being the airway epithelium. The effects of blocking TGF-β1 signaling had differential effects on AHR, fibrosis, and inflammation. While TGF-β neutralization may be beneficial to abrogating airway remodeling, it may be detrimental to lung function by increasing AHR.
lung; mice; hypersensitivity; cytokines