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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.

Muscle wasting is often associated with chronic inflammation. Because tumor necrosis factor alpha (TNF-alpha) has been implicated as a major mediator of cachexia, its effects on C2C12 myocytes were examined. TNF-alpha activated nuclear factor-kappaB (NF-kappaB) and interfered with the expression of muscle proteins in differentiating myoblasts. Introduction of a mutant form of inhibitory protein kappaBalpha (IkappaBalpha) restored myogenic differentiation in myoblasts treated with TNF-alpha or interleukin 1beta. Conversely, activation of NF-kappaB by overexpression of IkappaB kinase was sufficient to block myogenesis, illustrating the causal link between NF-kappaB activation and inhibition of myogenic differentiation. The inhibitory effects of TNF-alpha on myogenic differentiation were reversible, indicating that the effects of the cytokine were not due to nonspecific toxicity. Treatment of differentiated myotubes with TNF-alpha did not result in a striking loss of muscle-specific proteins, which shows that myogenesis was selectively affected in the myoblast stage by TNF-alpha. An important finding was that NF-kappaB was activated to the same extent in differentiating and differentiated cells, illustrating that once myocytes have differentiated they become refractory to the effects of NF-kappaB activation. These results demonstrate that inflammatory cytokines may contribute to muscle wasting through the inhibition of myogenic differentiation via a NF-kappaB-dependent pathway.

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.

Tumor necrosis factor alpha (TNFalpha) has been implicated as a mediator of muscle wasting through nuclear factor kappa B (NF-kappaB) -dependent inhibition of myogenic differentiation. The aim of the present study was to identify the regulatory molecule(s) of myogenesis targeted by TNFalpha/NF-kappaB signaling. TNFalpha interfered with cell cycle exit and repressed the accumulation of transcripts encoding muscle-specific genes in differentiating C2C12 myoblasts. Overexpression of a p65 (RelA) mutant lacking the transcriptional activation domain attenuated the TNFalpha-mediated inhibition of muscle-specific gene transcription. The ability of muscle regulatory factor MyoD to induce muscle-specific transcription in 10T1/2 fibroblasts was also disrupted by wild-type p65, demonstrating that NF-kappaB transcriptional activity interferes with the function of MyoD. Inhibition of muscle-specific gene expression by TNFalpha was restored by overexpression of MyoD, whereas endogenous MyoD protein abundance and stability were reduced by TNFalpha through increased proteolysis of MyoD by the ubiquitin proteasome pathway. Last, the inhibitory effects of TNFalpha on myogenic differentiation were demonstrated in a mouse model of skeletal muscle regeneration, in which TNFalpha caused a delay in myoblast cell cycle exit. These results implicate that TNFalpha inhibits myogenic differentiation through destabilizing MyoD protein in a NF-kappaB-dependent manner, which interferes with skeletal muscle regeneration and may contribute to muscle wasting.

Background The interleukin (IL)-1 family member IL-33 plays a critical role in type-2 innate immune responses to allergens, and is an important mediator of allergic asthma. The mechanisms by which allergens provoke epithelial IL-33 secretion are still poorly understood. Objective Based on previous findings indicating involvement of the NADPH oxidase DUOX1 in epithelial wound responses, we explored the potential involvement of DUOX1 in allergen-induced IL-33 secretion and potential alterations in airways of subjects with asthma. Methods Cultured human or murine airway epithelial cells or mice were subjected to acute challenge with Alternaria alternata or house dust mite (HDM), and secretion of IL-33 and activation of subsequent type 2 responses were determined. The role of DUOX1 was explored using siRNA approaches and DUOX1-deficient mice. Cultured nasal epithelial cells from healthy or asthmatic subjects were evaluated for DUOX1 expression and allergen-induced responses. Results In vitro or in vivo allergen challenge resulted in rapid airway epithelial IL-33 secretion, which critically depended on DUOX1-mediated activation of epithelial epidermal growth factor receptor (EGFR) and the protease calpain-2, via a redox-dependent mechanism involving cysteine oxidation within EGFR and the tyrosine kinase Src. Primary nasal epithelial cells from subjects with allergic asthma were found to express elevated DUOX1 and IL-33, and demonstrated enhanced IL-33 secretion in response to allergen challenge compared to nasal epithelial cells from non-asthmatic subjects. Conclusion Our findings implicate epithelial DUOX1 as a pivotal mediator of IL-33-dependent activation of innate airway type 2 immune responses to common airborne allergens, and indicate that enhanced DUOX1 expression and IL-33 secretion may present important contributing features of allergic asthma.

NF-kappaB is an inducible transcription factor that plays a role in the expression of over one hundred genes involved in immunity, inflammation, proliferation, and in defense against apoptosis. NF-kappaB has been known to be redox regulated for some time and is a direct target for oxidation that can affect its ability to bind to DNA. Reactive oxygen species (ROS) have been identified as second messengers in cells, and play a role in receptor signaling and posttranslation modification of signaling molecules. These posttranslation modifications include oxidations of critical cysteines to sulfenic acids or mixed disulfides, which can affect the activity of proteins. Many kinases involved in direct or indirect activation of NF-kappaB are affected by oxidants and therefore, have the potential to alter NF-kappaB activity. This review will provide a summary of the NF-kappaB family, their activation and regulation, followed by a summary of cytoplasmic and nuclear kinases in this pathway whose activity is affected by oxidants. Additionally, recent investigations have revealed that the JNK signaling pathway, which is known to be redox regulated, and pro-apoptotic, is inhibited by NF-kappaB signaling. The crosstalk of NF-kappaB with other signaling pathways is therefore critical for cellular fate, notably survival or cell death under oxidative conditions, and will also be reviewed.