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1.  Cigarette Smoke Targets Glutaredoxin 1, Increasing S-Glutathionylation and Epithelial Cell Death 
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.
PMCID: PMC3262689  PMID: 21454804
chronic obstructive pulmonary disease; cigarette smoke; cell death; glutaredoxin; protein S-glutathionylation
2.  Activation of the glutaredoxin-1 gene by Nuclear Factor kappa B enhances signaling 
Free radical biology & medicine  2011;51(6):1249-1257.
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.
PMCID: PMC3181077  PMID: 21762778
S-glutathionylation; Nuclear Factor kappa B; Glutaredoxin; Lung; Inhibitory kappa B kinase
3.  Altered Cigarette Smoke-Induced Lung Inflammation Due to Ablation of Grx1 
PLoS ONE  2012;7(6):e38984.
Glutaredoxins (Grx) are redox enzymes that remove glutathione bound to protein thiols, know as S-glutathionylation (PSSG). PSSG is a reservoir of GSH and can affect the function of proteins. It inhibits the NF-κB pathway and LPS aspiration in Grx1 KO mice with decreased inflammatory cytokine levels. In this study we investigated whether absence of Grx1 similarly repressed cigarette smoke-induced inflammation in an exposure model in mice. Cigarette smoke exposure for four weeks decreased lung PSSG levels, but increased PSSG in lavaged cells and lavage fluid (BALF). Grx1 KO mice had increased levels of PSSG in lung tissue, BALF and BAL cells in response to smoke compared to wt mice. Importantly, levels of multiple inflammatory mediators in the BALF were decreased in Grx1 KO animals following cigarette smoke exposure compared to wt mice, as were levels of neutrophils, dendritic cells and lymphocytes. On the other hand, macrophage numbers were higher in Grx1 KO mice in response to smoke. Although cigarette smoke in vivo caused inverse effects in inflammatory and resident cells with respect to PSSG, primary macrophages and epithelial cells cultured from Grx1 KO mice both produced less KC compared to cells isolated from WT mice after smoke extract exposure. In this manuscript, we provide evidence that Grx1 has an important role in regulating cigarette smoke-induced lung inflammation which seems to diverge from its effects on total PSSG. Secondly, these data expose the differential effect of cigarette smoke on PSSG in inflammatory versus resident lung cells.
PMCID: PMC3377591  PMID: 22723915
4.  Ablation of Glutaredoxin-1 Attenuates Lipopolysaccharide-Induced Lung Inflammation and Alveolar Macrophage Activation 
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.
PMCID: PMC3095922  PMID: 20539014
lipopolysaccharide; glutaredoxin-1; protein S-glutathionylation; nuclear factor-κB
Methods in enzymology  2010;474:289-296.
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.
PMCID: PMC3113509  PMID: 20609917
6.  Regulation of apoptosis through cysteine oxidation: implications for fibrotic lung disease 
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.
PMCID: PMC2943339  PMID: 20716279
S-glutathionylation; glutaredoxin; Fas; apoptosis; fibrosis; lung
7.  Nuclear Factor κB, Airway Epithelium, and Asthma 
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.
PMCID: PMC2677399  PMID: 19387025
epithelium; nuclear factor κB; asthma; oxidants
8.  Catalase Overexpression Fails to Attenuate Allergic Airways Disease in the Mouse1 
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.
PMCID: PMC2830272  PMID: 17339480
9.  Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas 
The Journal of Cell Biology  2009;184(2):241-252.
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.
PMCID: PMC2654302  PMID: 19171757

Results 1-9 (9)