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

Distinct Functions of Airway Epithelial Nuclear Factor-κB Activity Regulate Nitrogen Dioxide–Induced Acute Lung Injury

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

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

c-Jun N-Terminal Kinase 1 Promotes Transforming Growth Factor–β1–Induced Epithelial-to-Mesenchymal Transition via Control of Linker Phosphorylation and Transcriptional Activity of Smad3

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

Nitric oxide represses inhibitory κB kinase through S-nitrosylation

Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-κB. A commonly proposed mode of action for inhibition of NF-κBbyNO involves interference with NF-κB binding to DNA. Because activation of inhibitory κB kinase (IKK), the prerequisite enzyme complex necessary to induce NF-κB, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-κB activation. We demonstrate here that S-nitrosothiols (SNO) caused a dose-dependent inhibition of the enzymatic activity of IKK, in lung epithelial cells and in Jurkat T cells, which was associated with S-nitrosylation of the IKK complex. Using biotin derivatization of SNO, we revealed that IKKβ, the catalytic subunit required for NF-κB activation, was a direct target for S-nitrosylation. A mutant version of IKKβ containing a Cys-179-to-Ala mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKKβ, demonstrating that Cys-179 is the main target for attack by SNO. Importantly, inhibition of NO synthase activity in Jurkat T cells resulted in activation of IKK, in association with its denitrosylation. Moreover, NO synthase inhibition enhanced the ability of tumor necrosis factor α to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-κB activation by cytokines. Collectively, our findings demonstrate that IKKβ is an important target for the redox regulation of NF-κB by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties

Strain-dependent activation of NF-κB in the airway epithelium and its role in allergic airway inflammation

NF-κB activation in the airway epithelium has been established as a critical pathway in ovalbumin (Ova)-induced airway inflammation in BALB/c mice (Poynter ME, Cloots R, van Woerkom T, Butnor KJ, Vacek P, Taatjes DJ, Irvin CG, Janssen-Heininger YM. J Immunol 173: 7003–7009, 2004). BALB/c mice are susceptible to the development of allergic airway disease, whereas other strains of mice, such as C57BL/6, are considered more resistant. The goal of the present study was to determine the proximal signals required for NF-κB activation in the airway epithelium in allergic airway disease and to unravel whether these signals are strain-dependent. Our previous studies, conducted in the BALB/c mouse background, demonstrated that transgenic mice expressing a dominant-negative version of IκBα in the airway epithelium (CC10-IκBαSR) were protected from Ova-induced inflammation. In contrast to these earlier observations, we demonstrate here that CC10-IκBαSR transgenic mice on the C57BL/6 background were not protected from Ova-induced allergic airway inflammation. Consistent with this finding, Ova-induced nuclear localization of the RelA subunit of NF-κB was not observed in C57BL/6 mice, in contrast to the marked nuclear presence of RelA in BALB/c mice. Evaluation of cytokine profiles in bronchoalveolar lavage demonstrated elevated expression of TNF-α in BALB/c mice compared with C57BL/6 mice after an acute challenge with Ova. Finally, neutralization of TNF-α by a blocking antibody prevented nuclear localization of RelA in BALB/c mice after Ova challenge. These data suggest that the mechanism of response of the airway epithelium of immunized C57BL/6 mice to antigen challenge is fundamentally different from that of immunized BALB/c mice and highlight the potential importance of TNF-α in regulating epithelial NF-κB activation in allergic airway disease