The transcription factor NF-B, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory B kinase (IKK) ␤-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-␤ Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity.

Nitric oxide (NO) possesses antiinflammatory effects, which may be exerted via its ability to inhibit the transcription factor, NF-B. A commonly proposed mode of action for inhibition of NF-B by NO involves interference with NF-B binding to DNA. Because activation of inhibitory B kinase (IKK), the prerequisite enzyme complex necessary to induce NF-B, is subject to redox regulation, we assessed whether IKK could present a more proximal target for NO to inhibit NF-B 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 IKK␤, the catalytic subunit required for NF-B activation, was a direct target for S-nitrosylation. A mutant version of IKK␤ containing a Cys-179-toAla mutation was refractory to inhibition by SNO or to increases in S-nitrosylation, in contrast to wild-type IKK␤, 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 ␣ to activate IKK, illustrating the importance of endogenous NO in regulating the extent of NF-B activation by cytokines. Collectively, our findings demonstrate that IKK␤ is an important target for the redox regulation of NF-B by endogenous or exogenous NO, providing an additional mechanism for its antiinflammatory properties.

Rationale: Nuclear factor (NF)-kB is a prominent proinflammatory transcription factor that plays a critical role in allergic airway disease. Previous studies demonstrated that inhibition of NF-kB in airway epithelium causes attenuation of allergic inflammation. Objectives: We sought to determine if selective activation of NF-kB within the airway epithelium in the absence of other agonists is sufficient to cause allergic airway disease. Methods: A transgenic mouse expressing a doxycycline (Dox)-inducible, constitutively active (CA) version of inhibitor of kB (IkB) kinase-b (IKKb) under transcriptional control of the rat CC10 promoter, was generated. Measurements and Main Results: After administration of Dox, expression of the CA-IKKb transgene induced the nuclear translocation of RelA in airway epithelium. IKKb-triggered activation of NF-kB led to an increased content of neutrophils and lymphocytes, and concomitant production of proinflammatory mediators, responses that were not observed in transgenic mice not receiving Dox, or in transgenenegative littermate control animals fed Dox. Unexpectedly, expression of the IKKb transgene in airway epithelium was sufficient to cause airway hyperresponsiveness and smooth muscle thickening in absence of an antigen sensitization and challenge regimen, the presence of eosinophils, or the induction of mucus metaplasia.Conclusions: These findings demonstrate that selective activation NFkB in airway epithelium is sufficient to induce airway hyperresponsiveness and smooth muscle thickening, which are both critical features of allergic airway disease.

Transforming growth factor β1 (TGF-β1) is a cardinal cytokine in the pathogenesis of airway remodeling, and promotes epithelial-to-mesenchymal transition (EMT). As a molecular interaction between TGF-β1 and Jun N-terminal kinase (JNK) has been demonstrated, the goal of this study was to elucidate whether JNK plays a role in TGF-β1-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-β1. Wild-type MTEC exposed to TGF-β1 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-β1-mediated EMT was markedly blunted in epithelial cells lacking JNK1, while JNK2–/– MTEC underwent EMT in response to TGF-β1 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-β1, Smad DNA-binding activity was diminished. Gene expression profiling demonstrated a global suppression of TGF-β1-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.