Regulation of NF-kappaB in Lung Epithelium by ROS/RNS

University Of Vermont & St Agric College, Burlington VT

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Abstract

[unreadable] DESCRIPTION (provided by applicant): The transcription factor, Nuclear Factor kappa B (NF-?B) plays a pivotal role in inflammation and local activation of NF-?B in the airway epithelium plays a crucial role in the causation of airway inflammation. NF-?B is redox sensitive, and changes in the oxidative environment are critical in controlling its activation. During the current funding period we demonstrated that hydrogen peroxide (H2O2), represses the activation of NF-?B through reversible oxidation of a specific cysteine residue within Inhibitory kappa B kinase-beta (IKK[unreadable]), the prerequisite kinase driving NF-?B activation. S-glutathionylation of Cysteine 179 within IKK[unreadable] is the critical oxidation responsible for IKK inactivation by H2O2. S-glutathionylation has emerged as a newly recognized reversible oxidation relevant to signal transduction, since glutaredoxin (GRX) enzymes serve to specifically reduce S-glutathionylated cysteines, regenerating the reduced sulfhydryl group. We have demonstrated using overexpression or SiRNA approaches that glutaredoxin-1 (GRX1) is critical in controlling the magnitude of NF-?B activation. Since redox changes accompany the pathophysiology of allergic airways disease, and GRX1 is upregulated in airway epithelium of mice with allergic airways disease, GRX1 has the potential to contribute to the chronic NF-?B activation seen in mice with allergic inflammation. The hypothesis of the current proposal is that S-glutathionylation perturbs the IKK[unreadable] signalsome to repress activation of NF-?B. Glutaredoxin-1 (GRX1), by reversing S-glutathionylated cysteines overcomes this oxidative inhibition, and promotes NF-?B activation in the lung, leading to aggravated inflammation. Specific Aim #1 will elucidate biochemical basis for IKK[unreadable] inhibition by S- glutathionylation through examining IKK signalsome organization, and interactions with GRX1. We also will explore the interplay between S-glutathionylation of Cysteine 179, and phosphorylation of flanking Serines 177 and 181 of IKK[unreadable][unreadable]. Specific Aim #2 will determine whether changes in steady state levels of S- glutathionylation following modulation of Glutaredoxin-1 (GRX1) affect NF-?B activation and inflammatory gene expression in primary mouse tracheal epithelial cells in response to pro-inflammatory stimuli. Specific Aim #3 will determine whether mice lacking Glutaredoxin-1 (GRX1-/-) display enhanced protein-S- glutathionylation, lowered activation of NF-?B, and inflammation compared to wildtype mice, in response to sensitization and challenge with Ovalbumin (Ova). Specific Aim #4 will determine whether conditional overexpression of glutaredoxin-1 (GRX1) in mice lowers protein S-glutathionylation, and enhances activation of NF-?B, and subsequent inflammation induced by Ova. These approaches will provide critical insights into the physiological relevance of GRX1-controlled redox regulation of NF-?B in airways. [unreadable] [unreadable] [unreadable]