ERK-dependent proteasome degradation of Txnip regulates thioredoxin oxidoreductase activity.

Dynamic control of thioredoxin (Trx) oxidoreductase activity is essential for balancing the need of cells to rapidly respond to oxidative/nitrosative stress and to temporally regulate thiol-based redox signaling. We have previously shown that cytokine stimulation of the respiratory epithelium induces a precipitous decline in cell S-nitrosothiol, which depends upon enhanced Trx activity and proteasome-mediated degradation of Txnip (thioredoxin-interacting protein). We now show that tumor necrosis factor-α-induced Txnip degradation in A549 respiratory epithelial cells is regulated by the extracellular signal-regulated protein kinase (ERK) mitogen-activated protein kinase pathway and that ERK inhibition augments both intracellular reactive oxygen species and S-nitrosothiol. ERK-dependent Txnip ubiquitination and proteasome degradation depended upon phosphorylation of a PXTP motif threonine (Thr349) located within the C-terminal α-arrestin domain and proximal to a previously characterized E3 ubiquitin ligase-binding site. Collectively, these findings demonstrate the ERK mitogen-activated protein kinase pathway to be integrally involved in regulating Trx oxidoreductase activity and that the regulation of Txnip lifetime via ERK-dependent phosphorylation is an important mediator of this effect.

Thioredoxin-mediated denitrosylation regulates cytokine-induced nuclear factor κB (NF-κB) activation.

S-nitrosylation of nuclear factor κB (NF-κB) on the p65 subunit of the p50/p65 heterodimer inhibits NF-κB DNA binding activity. We have recently shown that p65 is constitutively S-nitrosylated in the lung and that LPS-induced injury elicits a decrease in SNO-p65 levels concomitant with NF-κB activation in the respiratory epithelium and initiation of the inflammatory response. Here, we demonstrate that TNFα-mediated activation of NF-κB in the respiratory epithelium similarly induces p65 denitrosylation. This process is mediated by the denitrosylase thioredoxin (Trx), which becomes activated upon cytokine-induced degradation of thioredoxin-interacting protein (Txnip). Similarly, inhibition of Trx activity in the lung attenuates LPS-induced SNO-p65 denitrosylation, NF-κB activation, and airway inflammation, supporting a pathophysiological role for this mechanism in lung injury. These data thus link stimulus-coupled activation of NF-κB to a specific, protein-targeted denitrosylation mechanism and further highlight the importance of S-nitrosylation in the regulation of the immune response.

S-nitrosylation of Ras in breast cancer.

Elevated expression of nitric oxide synthase 2 has been recently shown to correlate with poor survival in estrogen receptor-negative breast cancer. In an article in Breast Cancer Research, Switzer and colleagues identify the transcription factor Ets-1 as a critical mediator of nitric oxide-dependent oncogenic gene expression in basal-like breast cancer. This pathway is driven by S-nitrosylation of wild-type Ras, which leads to mitogen-activated protein kinase-dependent phosphorylation and activation of Ets-1. These results establish a new role for S-nitrosylation in mediating an aggressive breast cancer phenotype.

The clinical and environmental determinants of airway transcriptional profiles in allergic asthma.

RATIONALE: Gene expression profiling of airway epithelial and inflammatory cells can be used to identify genes involved in environmental asthma. METHODS: Airway epithelia and inflammatory cells were obtained via bronchial brush and bronchoalveolar lavage (BAL) from 39 subjects comprising three phenotypic groups (nonatopic nonasthmatic, atopic nonasthmatic, and atopic asthmatic) 4 hours after instillation of LPS, house dust mite antigen, and saline in three distinct subsegmental bronchi. RNA transcript levels were assessed using whole genome microarrays. MEASUREMENTS AND MAIN RESULTS: Baseline (saline exposure) differences in gene expression were related to airflow obstruction in epithelial cells (C3, ALOX5AP, CCL18, and others), and to serum IgE (innate immune genes and focal adhesion pathway) and allergic-asthmatic phenotype (complement genes, histone deacetylases, and GATA1 transcription factor) in inflammatory cells. LPS stimulation resulted in pronounced transcriptional response across all subjects in both airway epithelia and BAL cells, with strong association to nuclear factor-κB and IFN-inducible genes as well as signatures of other transcription factors (NRF2, C/EBP, and E2F1) and histone proteins. No distinct transcriptional profile to LPS was observed in the asthma and atopy phenotype. Finally, although no consistent expression changes were observed across all subjects in response to house dust mite antigen stimulation, we observed subtle differences in gene expression (e.g., GATA1 and GATA2) in BAL cells related to the asthma and atopy phenotype. CONCLUSIONS: Our results indicate that among individuals with allergic asthma, transcriptional changes in airway epithelia and inflammatory cells are influenced by phenotype as well as environmental exposures.

S-nitrosylation in the regulation of gene transcription.

BACKGROUND: Post-translational modification of proteins by S-nitrosylation serves as a major mode of signaling in mammalian cells and a growing body of evidence has shown that transcription factors and their activating pathways are primary targets. S-nitrosylation directly modifies a number of transcription factors, including NF-κB, HIF-1, and AP-1. In addition, S-nitrosylation can indirectly regulate gene transcription by modulating other cell signaling pathways, in particular JNK kinase and ras. SCOPE OF REVIEW: The evolution of S-nitrosylation as a signaling mechanism in the regulation of gene transcription, physiological advantages of protein S-nitrosylation in the control of gene transcription, and discussion of the many transcriptional proteins modulated by S-nitrosylation is summarized. MAJOR CONCLUSIONS: S-nitrosylation plays a crucial role in the control of mammalian gene transcription with numerous transcription factors regulated by this modification. Many of these proteins serve as immunomodulators, and inducible nitric oxide synthase (iNOS) is regarded as a principal mediatiator of NO-dependent S-nitrosylation. However, additional targets within the nucleus (e.g. histone deacetylases) and alternative mechanisms of S-nitrosylation (e.g. GAPDH-mediated trans-nitrosylation) are thought to play a role in NOS-dependent transcriptional regulation. GENERAL SIGNIFICANCE: Derangement of SNO-regulated gene transcription is an important factor in a variety of pathological conditions including neoplasia and sepsis. A better understanding of protein S-nitrosylation as it relates to gene transcription and the physiological mechanisms behind this process is likely to lead to novel therapies for these disorders. This article is part of a Special Issue entitled Regulation of Cellular Processes by S-nitrosylation.

NOS2 regulation of LPS-induced airway inflammation via S-nitrosylation of NF-{kappa}B p65.

Inducible nitric oxide synthase (NOS2) expression is increased in the airway epithelium in acute inflammatory disorders although the physiological impact remains unclear. We have previously shown that NOS2 inhibits NF-κB (p50-p65) activation in respiratory epithelial cells by inducing S-nitrosylation of the p65 monomer (SNO-p65). In addition, we have demonstrated that mouse lung SNO-p65 levels are acutely depleted in a lipopolysaccharide (LPS) model of lung injury and that augmenting SNO-p65 levels before LPS treatment results in decreased airway epithelial NF-κB activation, airway inflammation, and lung injury. We now show that aerosolized LPS induces NOS2 expression in the respiratory epithelium concomitant with an increase in lung SNO-p65 levels and a decrease in airway NF-κB activity. Genetic deletion of NOS2 results in an absence of SNO-p65 formation, persistent NF-κB activity in the respiratory epithelium, and prolonged airway inflammation. These results indicate that a primary function of LPS-induced NOS2 expression in the respiratory epithelium is to modulate the inflammatory response through deactivation of NF-κB via S-nitrosylation of p65, thereby counteracting the initial stimulus-coupled denitrosylation.

Protection from lipopolysaccharide-induced lung injury by augmentation of airway S-nitrosothiols.

RATIONALE: S-Nitrosothiols (SNO) inhibit immune activation of the respiratory epithelium and airway SNO levels are decreased in inflammatory lung disease. Ethyl nitrite (ENO) is a gas with chemical properties favoring SNO formation. Augmentation of airway SNO by inhaled ENO treatment may decrease lung inflammation and subsequent injury by inhibiting activation of the airway epithelium. OBJECTIVES: To determine the effect of inhaled ENO on airway SNO levels and LPS-induced lung inflammation/injury. METHODS: Mice were treated overnight with inhaled ENO (10 ppm) or air, followed immediately by exposure to aerosolized LPS or saline. Parameters of inflammation and lung injury were quantified 1 hour after completion of the aerosol exposure and correlated to lung airway and tissue SNO levels. MEASUREMENTS AND MAIN RESULTS: Aerosolized LPS induced a decrease in airway and lung tissue SNO levels including S-nitrosylated NF-kappaB. The decrease in lung SNO was associated with an increase in lung NF-kappaB activity, cytokine/chemokine expression (keratinocyte-derived chemokine, tumor necrosis factor-alpha, and IL-6), airway neutrophil influx, and worsened lung compliance. Pretreatment with inhaled ENO restored airway SNO levels and reduced LPS-mediated NF-kappaB activation thereby inhibiting the downstream inflammatory response and preserving lung compliance. CONCLUSIONS: Airway SNO serves an antiinflammatory role in the lung. Inhaled ENO can be used to augment airway SNO and protect from LPS-induced acute lung injury.

NOS2 regulation of NF-kappaB by S-nitrosylation of p65.

Signal transduction in the NF-kappaB transcription factor pathway is inhibited by inducible nitric oxide synthase (NOS2) activity, although the molecular mechanism(s) are incompletely understood. We have previously shown that nitric oxide (NO), derived from NOS2 consequent upon cytokine stimulation, attenuates NF-kappaB p50-p65 heterodimer DNA binding and have identified the p50 monomer as a locus for inhibitory S-nitrosylation. We now show that the binding partner of p50, NF-kappaB p65, is also targeted by NO following cytokine stimulation of respiratory epithelial cells and macrophages and identify a conserved cysteine within the Rel homology domain that is the site for S-nitrosylation. S-Nitrosylation of p65 inhibits NF-kappaB-dependent gene transcription, and nuclear levels of S-nitrosylated p65 correlate with decreased DNA binding of the p50-p65 heterodimer. NOS2 regulates cytokine-induced S-nitrosylation of p65, resulting in decreased NF-kappaB binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-kappaB activity and regulates gene expression in inflammation.

Protein S-nitrosylation: purview and parameters.

S-nitrosylation, the covalent attachment of a nitrogen monoxide group to the thiol side chain of cysteine, has emerged as an important mechanism for dynamic, post-translational regulation of most or all main classes of protein. S-nitrosylation thereby conveys a large part of the ubiquitous influence of nitric oxide (NO) on cellular signal transduction, and provides a mechanism for redox-based physiological regulation.

Essential roles of S-nitrosothiols in vascular homeostasis and endotoxic shock.

The current perspective of NO biology is formulated predominantly from studies of NO synthesis. The role of S-nitrosothiol (SNO) formation and turnover in governing NO-related bioactivity remains uncertain. We generated mice with a targeted gene deletion of S-nitrosoglutathione reductase (GSNOR), and show that they exhibit substantial increases in whole-cell S-nitrosylation, tissue damage, and mortality following endotoxic or bacterial challenge. Further, GSNOR(-/-) mice have increased basal levels of SNOs in red blood cells and are hypotensive under anesthesia. Thus, SNOs regulate innate immune and vascular function, and are cleared actively to ameliorate nitrosative stress. Nitrosylation of cysteine thiols is a critical mechanism of NO function in both health and disease.

Nitrosative stress-induced apoptosis through inhibition of NF-kappa B.

Nitrosative stress produced by cytokines predisposes to apoptotic cell death. However, the molecular mechanism by which this occurs is not well understood. We have shown previously that nitric oxide (NO) regulates the activity of the anti-apoptotic transcription factor NF-kappaB. Here we demonstrate that the inhibition of NF-kappaB by NO sensitizes A549 and Jurkat T cells to tumor necrosis factor-alpha (TNFalpha)-induced apoptosis. The molecular basis of NF-kappaB inhibition is different in the two cell types. In A549 cells, NO functions at the nuclear level to inhibit NF-kappaB by S-nitrosylation. In Jurkat cells, NO inhibits the NF-kappaB activating pathway in the cytoplasm at a step proximal to the degradation of IkappaBalpha. The inhibition of NF-kappaB is reflected in the level of intracellular S-nitrosothiols, which are constitutively metabolized. These data suggest that NO can influence cell death by modulating NF-kappaB activity with the sites of inhibition being cell type-specific. The data also show that NO bioactivity regulates tumor necrosis factor-alpha signaling.