Publications (15)83.74 Total impact
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Article: Emerging Mechanisms of Glutathione-dependent Chemistry in Biology and Disease.
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ABSTRACT: Glutathione has traditionally been considered as an antioxidant that protects cells against oxidative stress. Hence, the loss of reduced glutathione and formation of glutathione disulfide is considered a classical parameter of oxidative stress that is increased in diseases. Recent studies have emerged that demonstrate that glutathione plays a more direct role in biological and pathophysiological processes through covalent modification to reactive cysteines within proteins, a process known as S-glutathionylation. The formation of an S-glutathionylated moiety within the protein can lead to structural and functional modifications. Activation, inactivation, loss of function, and gain of function have all been attributed to S-glutathionylation. In pathophysiological settings, S-glutathionylation is tightly regulated. This perspective offers a concise overview of the emerging field of protein thiol redox modifications. We will also cover newly developed methodology to detect S-glutathionylation in situ, which will enable further discovery into the role of S-glutathionylation in biology and disease. J. Cell. Biochem. © 2013 Wiley Periodicals, Inc.Journal of Cellular Biochemistry 03/2013; · 2.87 Impact Factor -
Article: Oxidative processing of latent Fas in the endoplasmic reticulum controls the strength of apoptosis.
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ABSTRACT: 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.Molecular and cellular biology 07/2012; 32(17):3464-78. · 6.06 Impact Factor -
Article: Genetic ablation of glutaredoxin-1 causes enhanced resolution of airways hyperresponsiveness and mucus metaplasia in mice with allergic airways disease.
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ABSTRACT: Protein-S-glutathionylation (PSSG) is an oxidative modification of reactive cysteines that has emerged as an important player in pathophysiological processes. Under physiological conditions, the thiol transferase, glutaredoxin-1 (Glrx1) catalyses deglutathionylation. Although we previously demonstrated that Glrx1 expression is increased in mice with allergic inflammation, the impact of Glrx1/PSSG in the development of allergic airways disease remains unknown. In the present study we examined the impact of genetic ablation of Glrx1 in the pathogenesis of allergic inflammation and airway hyperresponsiveness (AHR) in mice. Glrx1(-/-) or WT mice were subjected to the antigen, ovalbumin (OVA), and parameters of allergic airways disease were evaluated 48 h after three challenges, and 48 h or 7 days after six challenges with aerosolized antigen. Although no clear increases in PSSG were observed in WT mice in response to OVA, marked increases were detected in lung tissue of mice lacking Glrx1 48 h following six antigen challenges. Inflammation and expression of proinflammatory mediators were decreased in Glrx1(-/-) mice, dependent on the time of analysis. WT and Glrx1(-/-) mice demonstrated comparable increases in AHR 48 h after three or six challenges with OVA. However, 7 days postcessation of six challenges, parameters of AHR in Glrx1(-/-) mice were resolved to control levels, accompanied by marked decreases in mucus metaplasia and expression of Muc5AC and GOB5. These results demonstrate that the Glrx1/S-glutathionylation redox status in mice is a critical regulator of AHR, suggesting that avenues to increase S-glutathionylation of specific target proteins may be beneficial to attenuate AHR.AJP Lung Cellular and Molecular Physiology 06/2012; 303(6):L528-38. · 3.66 Impact Factor -
Article: Cooperation between Classical and Alternative NF-κB Pathways Regulates Proinflammatory Responses in Epithelial Cells.
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ABSTRACT: 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.American Journal of Respiratory Cell and Molecular Biology 05/2012; 47(4):497-508. · 5.13 Impact Factor -
Article: Redox-based regulation of apoptosis: S-glutathionylation as a regulatory mechanism to control cell death.
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ABSTRACT: 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. Although redox-based changes have been documented in apoptosis and disease pathogenesis, the mechanistic details, whereby redox perturbations intersect with pathogenic processes, remain obscure. 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.Antioxidants & Redox Signaling 09/2011; 16(6):496-505. · 8.20 Impact Factor -
Article: Activation of the glutaredoxin-1 gene by nuclear factor κB enhances signaling.
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ABSTRACT: The transcription factor nuclear factor κB (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 catalyzes 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 that express a doxycycline-inducible constitutively active version of inhibitory κB kinase-β (CA-IKKβ) demonstrate elevated expression of Grx1. Transient transfection of CA-IKKβ also resulted in significant induction of Grx1. A 2-kb region of the 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 proinflammatory 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.Free radical biology & medicine 09/2011; 51(6):1249-57. · 5.42 Impact Factor -
Article: Influenza induces endoplasmic reticulum stress, caspase-12-dependent apoptosis, and c-Jun N-terminal kinase-mediated transforming growth factor-β release in lung epithelial cells.
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ABSTRACT: Influenza A virus (IAV) infection is known to induce endoplasmic reticulum (ER) stress, Fas-dependent apoptosis, and TGF-β production in a variety of cells. However, the relationship between these events in murine primary tracheal epithelial cells (MTECS), which are considered one of the primary sites of IAV infection and replication, is unclear. We show that IAV infection induced ER stress marker activating transcription factor-6 and endoplasmic reticulum protein 57-kD (ERp57), but not C/EBP homologous protein (CHOP). In contrast, the ER stress inducer thapsigargin (THP) increased CHOP. IAV infection activated caspases and apoptosis, independently of Fas and caspase-8, in MTECs. Instead, apoptosis was mediated by caspase-12. A decrease in ERp57 attenuated the IAV burden and decreased caspase-12 activation and apoptosis in epithelial cells. TGF-β production was enhanced in IAV-infected MTECs, compared with THP or staurosporine. IAV infection caused the activation of c-Jun N-terminal kinase (JNK). Furthermore, IAV-induced TGF-β production required the presence of JNK1, a finding that suggests a role for JNK1 in IAV-induced epithelial injury and subsequent TGF-β production. These novel findings suggest a potential mechanistic role for a distinct ER stress response induced by IAV, and a profibrogenic/repair response in contrast to other pharmacological inducers of ER stress. These responses may also have a potential role in acute lung injury, fibroproliferative acute respiratory distress syndrome, and the recently identified H1N1 influenza-induced exacerbations of chronic obstructive pulmonary disease (Wedzicha JA. Proc Am Thorac Soc 2004;1:115-120) and idiopathic pulmonary fibrosis (Umeda Y, et al. Int Med 2010;49:2333-2336).American Journal of Respiratory Cell and Molecular Biology 07/2011; 46(5):573-81. · 5.13 Impact Factor -
Article: Ablation of glutaredoxin-1 attenuates lipopolysaccharide-induced lung inflammation and alveolar macrophage activation.
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ABSTRACT: 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.American Journal of Respiratory Cell and Molecular Biology 04/2011; 44(4):491-9. · 5.13 Impact Factor -
Article: Regulation of apoptosis through cysteine oxidation: implications for fibrotic lung disease.
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ABSTRACT: 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.Annals of the New York Academy of Sciences 08/2010; 1203:23-8. · 3.15 Impact Factor -
Article: In situ analysis of protein S-glutathionylation in lung tissue using glutaredoxin-1-catalyzed cysteine derivatization.
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ABSTRACT: Protein S-glutathionylation (PSSG) is a posttranslational modification that involves the conjugation of the small antioxidant molecule glutathione to cysteine residues and is emerging as a critical mechanism of redox-based signaling. PSSG levels increase under conditions of oxidative stress and are controlled by glutaredoxins (Grx) that, under physiological conditions, preferentially deglutathionylate cysteines and restore sulfhydryls. Both the occurrence and distribution of PSSG in tissues is unknown because of the labile nature of this oxidative event and the lack of specific reagents. The goal of this study was to establish and validate a protocol that enables detection of PSSG in situ, using the property of Grx to deglutathionylate cysteines. Using Grx1-catalyzed cysteine derivatization, we evaluated PSSG content in mice subjected to various models of lung injury and fibrosis. In control mice, PSSG was detectable primarily in the airway epithelium and alveolar macrophages. Exposure of mice to NO(2) resulted in enhanced PSSG levels in parenchymal regions, while exposure to O(2) resulted in minor detectable changes. Finally, bleomycin exposure resulted in marked increases in PSSG reactivity both in the bronchial epithelium as well as in parenchymal regions. Taken together, these findings demonstrate that Grx1-based cysteine derivatization is a powerful technique to specifically detect patterns of PSSG expression in lungs, and will enable investigations into regional changes in PSSG content in a variety of diseases.American Journal Of Pathology 08/2009; 175(1):36-45. · 4.89 Impact Factor -
Article: Nuclear factor kappaB, airway epithelium, and asthma: avenues for redox control.
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ABSTRACT: 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 kappaB, 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 kappaB activation and inflammation, and discuss recent assays that have become available to probe the functional roles of oxidants in lung biology.Proceedings of the American Thoracic Society 06/2009; 6(3):249-55. -
Article: Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas.
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ABSTRACT: 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.The Journal of Cell Biology 02/2009; 184(2):241-52. · 10.26 Impact Factor -
Article: Nonphagocytic oxidase 1 causes death in lung epithelial cells via a TNF-RI-JNK signaling axis.
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ABSTRACT: Airway epithelial cells are simultaneously exposed to and produce cytokines and reactive oxygen species (ROS) in inflammatory settings. The signaling events and the physiologic outcomes of exposure to these inflammatory mediators remain to be elucidated. Previously we demonstrated that in cultured mouse lung epithelial cells exposed to bolus administration of H(2)O(2), TNF-alpha-induced NF-kappaB activity was inhibited, whereas c-Jun-N-terminal kinase (JNK) activation was enhanced via a mechanism involving TNF receptor-1 (TNF-RI). In this study we used the nonphagocytic NADPH oxidase (Nox1) to study the effects of endogenously produced ROS on a line of mouse alveolar type II epithelial cells. Nox1 expression and activation inhibited TNF-alpha-induced inhibitor of kappaB kinase (IKK), and NF-kappaB while promoting JNK activation and cell death. Nox1-induced JNK activation and cell death were attenuated through expression of a dominant-negative TNF-RI construct, implicating a role for TNF-RI in Nox1 signaling. Furthermore, Nox1 used the TNF-RI adaptor protein TNF-receptor-associated factor-2 (TRAF2), and the redox-regulated JNK MAP3K, apoptosis signal kinase-1 (ASK1), to activate JNK. In addition, ASK1 siRNA attenuated both Nox1-induced JNK activity and cell death. Collectively, these studies suggest a mechanism by which ROS produced in lung epithelial cells activate JNK and cause cell death using TNF-RI and the TRAF2-ASK1 signaling axis.American Journal of Respiratory Cell and Molecular Biology 05/2007; 36(4):473-9. · 5.13 Impact Factor -
Article: Oxidation state governs structural transitions in peroxiredoxin II that correlate with cell cycle arrest and recovery.
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ABSTRACT: Inactivation of eukaryotic 2-Cys peroxiredoxins (Prxs) by hyperoxidation has been proposed to promote accumulation of hydrogen peroxide (H2O2) for redox-dependent signaling events. We examined the oxidation and oligomeric states of PrxI and -II in epithelial cells during mitogenic signaling and in response to fluxes of H2O2. During normal mitogenic signaling, hyperoxidation of PrxI and -II was not detected. In contrast, H2O2-dependent cell cycle arrest was correlated with hyperoxidation of PrxII, which resulted in quantitative recruitment of approximately 66- and approximately 140-kD PrxII complexes into large filamentous oligomers. Expression of cyclin D1 and cell proliferation did not resume until PrxII-SO2H was reduced and native PrxII complexes were regenerated. Ectopic expression of PrxI or -II increased Prx-SO2H levels in response to oxidant exposure and failed to protect cells from arrest. We propose a model in which Prxs function as peroxide dosimeters in subcellular processes that involve redox cycling, with hyperoxidation controlling structural transitions that alert cells of perturbations in peroxide homeostasis.The Journal of Cell Biology 01/2007; 175(5):779-89. · 10.26 Impact Factor -
Article: Redox-dependent expression of cyclin D1 and cell proliferation by Nox1 in mouse lung epithelial cells.
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ABSTRACT: NADPH oxidases produce reactive oxygen species (ROS) that serve as co-stimulatory signals for cell proliferation. In mouse lung epithelial cells that express Nox1, Nox2, Nox4, p22(phox), p47(phox), p67(phox), and Noxo1, overexpression of Nox1 delayed cell cycle withdrawal by maintaining AP-1-dependent expression of cyclin D1 in low serum conditions. In cycling cells, the effects of Nox1 were dose dependent: levels of Nox1 that induced 3- to 10-fold increases in ROS promoted phosphorylation of ERK1/2 and expression of cyclin D1, whereas expression of Nox1 with Noxo1 and Noxa1 (or expression of Nox4 alone) that induced substantial increases in intracellular ROS inhibited cyclin D1 and proliferation. Catalase reversed the effects of Nox1 on cyclin D1 and cell proliferation. Diphenylene iodonium, an inhibitor of NADPH oxidase activity, did not affect dosedependent responses of ERK1/2 or Akt to serum, but markedly inhibited the sequential expression of c-Fos and Fra-1 required for induction of cyclin D1 during cell cycle re-entry. These results indicate that Nox1 stimulates cell proliferation in actively cycling cells by reducing the requirement for growth factors to maintain expression of cyclin D1, whereas during cell cycle re-entry, NADPH oxidase activity is required for transcriptional activation of Fos family genes during the immediate early gene response.Antioxidants and Redox Signaling 8(9-10):1447-59. · 8.46 Impact Factor
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2007–2013
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University of Vermont
- Department of Pathology
Burlington, VT, USA
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