Jun N-terminal kinase 1 regulates epithelial-to-mesenchymal transition induced by TGF-beta1

Transforming growth factor beta1 (TGF-beta1) is a cardinal cytokine in the pathogenesis of airway remodeling, and promotes epithelial-to-mesenchymal transition (EMT). As a molecular interaction between TGF-beta1 and Jun N-terminal kinase (JNK) has been demonstrated, the goal of this study was to elucidate whether JNK plays a role in TGF-beta1-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-beta1. Wild-type MTEC exposed to TGF-beta1 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-beta1-mediated EMT was markedly blunted in epithelial cells lacking JNK1, while JNK2-/- MTEC underwent EMT in response to TGF-beta1 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-beta1, Smad DNA-binding activity was diminished. Gene expression profiling demonstrated a global suppression of TGF-beta1-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

Redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and S-glutathionylation of Fas

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

Rust never sleeps: the continuing story of the Iron Bolt

Since 1981, Gordon Research Conferences have been held on the topic of Oxygen Radicals on a biennial basis, to highlight and discuss the latest cutting edge research in this area. Since the first meeting, one special feature of this conference has been the awarding of the so-called Iron Bolt, an award that started in jest but has gained increasing reputation over the years. Since no written documentation exists for this Iron Bolt award, this perspective serves to overview the history of this unusual award, and highlights various experiences of previous winners of this “prestigious” award and other interesting anecdotes

TGF-β1-induced deposition of provisional extracellular matrix by tracheal basal cells promotes epithelial-to-mesenchymal transition in a c-Jun NH2-terminal kinase-1-dependent manner

Epithelial cells have been suggested as potential drivers of lung fibrosis, although the epithelial-dependent pathways that promote fibrogenesis remain unknown. Extracellular matrix is increasingly recognized as an environment that can drive cellular responses in various pulmonary diseases. In this study, we demonstrate that transforming growth factor-β1 (TGF-β1)-stimulated mouse tracheal basal (MTB) cells produce provisional matrix proteins in vitro, which initiate mesenchymal changes in subsequently freshly plated MTB cells via Rho kinase-and c-Jun NH2-terminal kinase (JNK1)-dependent processes. Repopulation of decellularized lung scaffolds, derived from mice with bleomycin-induced fibrosis or from patients with idiopathic pulmonary fibrosis, with wild-type MTB cells resulted in a loss of epithelial gene expression and augmentation of mesenchymal gene expression compared with cells seeded into decellularized normal lungs. In contrast, Jnk1-/- basal cells seeded into fibrotic lung scaffolds retained a robust epithelial expression profile, failed to induce mesenchymal genes, and differentiated into club cell secretory protein-expressing cells. This new paradigm wherein TGF-β1-induced extracellular matrix derived from MTB cells activates a JNK1-dependent mesenchymal program, which impedes subsequent normal epithelial cell homeostasis, provides a plausible scenario of chronic aberrant epithelial repair, thought to be critical in lung fibrogenesis. This study identifies JNK1 as a possible target for inhibition in settings wherein reepithelialization is desired

The redox mechanism for vascular barrier dysfunction associated with metabolic disorders: Glutathionylation of Rac1 in endothelial cells

AbstractBackgroundOxidative stress is implicated in increased vascular permeability associated with metabolic disorders, but the underlying redox mechanism is poorly defined. S-glutathionylation, a stable adduct of glutathione with protein sulfhydryl, is a reversible oxidative modification of protein and is emerging as an important redox signaling paradigm in cardiovascular physiopathology. The present study determines the role of protein S-glutathionylation in metabolic stress-induced endothelial cell permeability.Methods and resultsIn endothelial cells isolated from patients with type-2 diabetes mellitus, protein S-glutathionylation level was increased. This change was also observed in aortic endothelium in ApoE deficient (ApoE-/-) mice fed on Western diet. Metabolic stress-induced protein S-glutathionylation in human aortic endothelial cells (HAEC) was positively correlated with elevated endothelial cell permeability, as reflected by disassembly of cell-cell adherens junctions and cortical actin structures. These impairments were reversed by adenoviral overexpression of a specific de-glutathionylation enzyme, glutaredoxin-1 in cultured HAECs. Consistently, transgenic overexpression of human Glrx-1 in ApoE-/- mice fed the Western diet attenuated endothelial protein S-glutathionylation, actin cytoskeletal disorganization, and vascular permeability in the aorta. Mechanistically, glutathionylation and inactivation of Rac1, a small RhoGPase, were associated with endothelial hyperpermeability caused by metabolic stress. Glutathionylation of Rac1 on cysteine 81 and 157 located adjacent to guanine nucleotide binding site was required for the metabolic stress to inhibit Rac1 activity and promote endothelial hyperpermeability.ConclusionsGlutathionylation and inactivation of Rac1 in endothelial cells represent a novel redox mechanism of vascular barrier dysfunction associated with metabolic disorders

In Situ Analysis of Protein S-Glutathionylation in Lung Tissue Using Glutaredoxin-1-Catalyzed Cysteine Derivatization

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 NO2 resulted in enhanced PSSG levels in parenchymal regions, while exposure to O2 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

Glutathione S-transferase pi modulates NF-κB activation and pro-inflammatory responses in lung epithelial cells

Nuclear Factor kappa B (NF-κB) is a transcription factor family critical in the activation of pro- inflammatory responses. The NF-κB pathway is regulated by oxidant-induced post-translational modifications. Protein S-glutathionylation, or the conjugation of the antioxidant molecule, glutathione to reactive cysteines inhibits the activity of inhibitory kappa B kinase beta (IKKβ), among other NF-κB proteins. Glutathione S-transferase Pi (GSTP) is an enzyme that has been shown to catalyze protein S-glutathionylation (PSSG) under conditions of oxidative stress. The objective of the present study was to determine whether GSTP regulates NF-κB signaling, S-glutathionylation of IKK, and subsequent pro-inflammatory signaling. We demonstrated that, in unstimulated cells, GSTP associated with the inhibitor of NF-κB, IκBα. However, exposure to LPS resulted in a rapid loss of association between IκBα and GSTP, and instead led to a protracted association between IKKβ and GSTP. LPS exposure also led to increases in the S-glutathionylation of IKKβ. SiRNA-mediated knockdown of GSTP decreased IKKβ-SSG, and enhanced NF-κB nuclear translocation, transcriptional activity, and pro-inflammatory cytokine production in response to lipopolysaccharide (LPS). TLK117, an isotype-selective inhibitor of GSTP, also enhanced LPS-induced NF-κB transcriptional activity and pro-inflammatory cytokine production, suggesting that the catalytic activity of GSTP is important in repressing NF-κB activation. Expression of both wild-type and catalytically-inactive Y7F mutant GSTP significantly attenuated LPS- or IKKβ-induced production of GM-CSF. These studies indicate a complex role for GSTP in modulating NF-κB, which may involve S-glutathionylation of IKK proteins, and interaction with NF-κB family members. Our findings suggest that targeting GSTP is a potential avenue for regulating the activity of this prominent pro-inflammatory and immunomodulatory transcription factor