Signal transduction in the NF-B 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-B 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-B 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-B-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-B binding to the NOS2 promoter, thereby inhibiting further NOS2 expression. Collectively, these findings delineate a mechanism by which NOS2 modulates NF-B activity and regulates gene expression in inflammation.The transcription factor NF-B controls the expression of many genes involved in the inflammatory response (1). One of these genes is the inducible nitric oxide synthase (NOS2) 2 whose activity impacts the cellular response to acute injury (2). The product of NOS2, nitric oxide (NO), is known to modulate NF-B activity at multiple steps in the signal transduction pathway (3). The primary molecular mechanism by which NO alters NF-B signaling is via S-nitrosylation, with several different NF-B proteins including IB kinase ␤ and p50 regulated by this post-translational modification (4, 5). Particularly, we have shown that p50 is S-nitrosylated under conditions of nitrosative stress and is associated with a decrease in NF-B (p50-p65) DNA binding (4). However, the physiological significance of S-nitrosylation of the NF-B p50-p65 heterodimer in the context of cytokine signaling and cellular NOS2 expression has not been established.NOS2 expression is dependent upon NF-B activation, with the cytokine-responsive B-binding site(s) identified in both the human and the murine NOS2 promoters (6, 7). Cytokinestimulated NOS2 activity, in turn, inhibits NF-B-dependent transcription, but the specific molecular target(s) of NOS2 in the NF-B pathway have not been elucidated (8). We have previously demonstrated that cytokine-induced NOS activity inhibits NF-B DNA binding in a reversible manner, a mechanism consistent with S-nitrosylation of the p50-p65 heterodimer (4). Moreover, evidence accumulated recently suggests a central role of S-nitrosylation by NOS2 in the regulation of inflammatory mediators (9, 10).In the past, the p50 monomer was felt to be the probable target for NOS2-mediated S-nitrosylation of the p50-p65 heterodimer. This rationale was based on the initial identification of a single redox-sensitive cysteine (Cys-62) located in the DNA-binding region of p50 (11). Interestingly, this cysteine ...

Background: S-nitrosylation inhibits mediators of the immune response including the transcription factor NF-B (p50/p65). Results: Exogenous and endogenous inhibitors of thioredoxin prevent p65 denitrosylation and downstream activation of NF-B. Conclusion: Thioredoxin activates inflammatory signaling through targeted denitrosylation of NF-B. Significance: Mechanisms that augment protein S-nitrosylation can be utilized to suppress the immune response.

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-kB. The decrease in lung SNO was associated with an increase in lung NF-kB activity, cytokine/chemokine expression (keratinocyte-derived chemokine, tumor necrosis factor-a, and IL-6), airway neutrophil influx, and worsened lung compliance. Pretreatment with inhaled ENO restored airway SNO levels and reduced LPS-mediated NF-kB 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.

We describe the microcell fusion transfer of 100-200 kb self-replicating circular human minichromosomes from human into mouse cells. This experimental approach is illustrated through the shutting of the latent 170 kb double-stranded DNA genome from the human herpesvirus, Epstein-Barr virus, into nonpermissive rodent cells. Using this interspecies transfer strategy, circular episomes carrying 95-105 kb of human DNA were successfully established at low copy number in mouse A9 cells. Selected episomes were stably maintained for 6 months, and unselected episomes were characterized by a 95% episomal retention per cell division. The establishment of a mouse artificial episomal chromosome system should facilitate evolutionary and therapeutic studies of large human DNA in rodent genetic backgrounds.