Yvonne M. W. Janssen-Heininger

Recent advances torwards understanding redox mechanisms in the activation of nuclear factor κb

The transcription factor, nuclear factor-kappaB (NF-kappaB) has been studied extensively due to its prominent role in the regulation of immune and inflammatory genes, apoptosis, and cell proliferation. It has been known for more that a decade that NF-kappaB is a redox-sensitive transcription factor. The contribution of redox regulation and the location of potential redox-sensitive sites within the NF-kappaB activation pathway are subject to intense debate due to many conflicting reports. Redox regulation of NF-kappaB has been extensively addressed in this journal and the reader is referred to two comprehensive reviews on the subject [1,2]. With the identification of signaling intermediates proximal to the degradation of the inhibitor, IkappaB, the number of potential redox-sensitive sites is rapidly increasing. The purpose of this review is to address recent insights into the NF-kappaB signaling cascades that are triggered by proinflammatory cytokines such as TNF-alpha and IL-1beta. In addition, the role of nitrogen monoxide (.NO) in the regulation of NF-kappaB will be reviewed. Opportunities for redox regulation that occur upstream of IkappaB-alpha degradation, as well as the potential for redox control of phosphorylation of NF-kappaB subunits, will be discussed. Redox-sensitive steps are likely to depend on the nature of the NF-kappaB activator, the type of reactive oxygen or nitrogen species involved, the selectivity of signaling pathways activated, as well as the cell type under investigation. Lastly, it is discussed how redox regulation of NF-kappaB activation is likely to involve multiple subcellular compartments.

REDOX-BASED REGULATION OF SIGNAL TRANSDUCTION: PRINCIPLES, PITFALLS, AND PROMISES

Oxidants are produced as a by-product of aerobic metabolism, and organisms ranging from prokaryotes to mammals have evolved with an elaborate and redundant complement of antioxidant defenses to confer protection against oxidative insults. Compelling data now exist demonstrating that oxidants are used in physiological settings as signaling molecules with important regulatory functions controlling cell division, migration, contraction, and mediator production. These physiological functions are carried out in an exquisitely regulated and compartmentalized manner by mild oxidants, through subtle oxidative events that involve targeted amino acids in proteins. The precise understanding of the physiological relevance of redox signal transduction has been hampered by the lack of specificity of reagents and the need for chemical derivatization to visualize reversible oxidations. In addition, it is difficult to measure these subtle oxidation events in vivo. This article reviews some of the recent findings that illuminate the significance of redox signaling and exciting future perspectives. We also attempt to highlight some of the current pitfalls and the approaches needed to advance this important area of biochemical and biomedical research.

Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor-kappaB.

Muscle wasting is often associated with chronic inflammation. Because tumor necrosis factor α (TNF‐α) has been implicated as a major mediator of cachexia, its effects on C2C12 myocytes were examined. TNF‐α activated nuclear factor‐κΒ (NF‐κΒ) and interfered with the expression of muscle proteins in differentiating myoblasts. Introduction of a mutant form of inhibitory protein κΒα (IκBα) restored myogenic differentiation in myoblasts treated with TNF‐α or interleukin 1β. Conversely, activation of NF‐KBby overexpression of IΚB kinase was sufficient to block myogenesis, illustrating the causal link between NF‐ΚB activation and inhibition of myogenic differentiation. The inhibitory effects of TNF‐α on myogenic differentiation were reversible, indicating that the effects of the cytokine were not due to nonspecific toxicity. Treatment of differentiated myotubes with TNF‐α did not result in a striking loss of muscle‐specific proteins, which shows that myogenesis was selectively affected in the myoblast stage by TNF‐α. An important finding was that NF‐ΚB 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‐ΚB activation. These results demonstrate that inflammatory cytokines may contribute to muscle wasting through the inhibition of myogenic differentiation via a NF‐κB‐dependent pathway.—Langen, R. C. J., Schols, A. M. W. J., Kelders, M. C. J. M., Wouters, E. F. M., Janssen‐Heininger, Y. M. W. Inflammatory cytokines inhibit myogenic differentiation through activation of nuclear factor‐KB. FASEB J. 15, 1169–1180 (2001)

Dynamic redox control of NF-kappaB through glutaredoxin-regulated S-glutathionylation of inhibitory kappaB kinase beta

The transcription factor NF-κB, a central regulator of immunity, is subject to regulation by redox changes. We now report that cysteine-179 of the inhibitory κB kinase (IKK) β-subunit of the IKK signalosome is a central target for oxidative inactivation by means of S-glutathionylation. S-glutathionylation of IKK-β Cys-179 is reversed by glutaredoxin (GRX), which restores kinase activity. Conversely, GRX1 knockdown sensitizes cells to oxidative inactivation of IKK-β and dampens TNF-α-induced IKK and NF-κB activation. Primary tracheal epithelial cells from Glrx1-deficient mice display reduced NF-κB DNA binding, RelA nuclear translocation, and MIP-2 (macrophage inflammatory protein 2) and keratinocyte-derived chemokine production in response to LPS. Collectively, these findings demonstrate the physiological relevance of the S-glutathionylation–GRX redox module in controlling the magnitude of activation of the NF-κB pathway.

Tumor necrosis factor-alpha inhibits myogenic differentiation through MyoD protein destabilization

Tumor necrosis factor α (TNFα) has been implicated as a mediator of muscle wasting through nuclear factor kappa B (NF‐ΚB) ‐dependent inhibition of myogenic differentiation. The aim of the present study was to identify the regulatory molecule(s) of myogenesis targeted by TNFα/NF‐κΒ signaling. TNFα 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 TNFα‐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‐KB transcriptional activity interferes with the function of MyoD. Inhibition of muscle‐specific gene expression by TNFα was restored by overexpression of MyoD, whereas endogenous MyoD protein abundance and stability were reduced by TNFα through increased proteolysis of MyoD by the ubiquitin proteasome pathway. Last, the inhibitory effects of TNFα on myogenic differentiation were demonstrated in a mouse model of skeletal muscle regeneration, in which TNFα caused a delay in myoblast cell cycle exit. These results implicate that TNFα inhibits myogenic differentiation through destabilizing MyoD protein in a NF‐κB‐dependent manner, which interferes with skeletal muscle regeneration and may contribute to muscle wasting.—Langen, R. C. J., van der Velden, J. L. J., Schols, A. M. W. J., Kelders, M. C. J. M., Wouters, E. F. M., Janssen‐Heininger, Y. M. W. Tumor necrosis factor‐alpha inhibits myogenic differentiation through MyoD protein destabilization. FASEB J. 18, 227–237 (2004)

A Prominent Role for Airway Epithelial NF-κB Activation in Lipopolysaccharide-Induced Airway Inflammation

To reveal the causal role of airway epithelial NF-κB activation in evoking airway inflammation, a transgenic mouse was created expressing a mutant version of the inhibitory protein I-κBα. This I-κBα superrepressor (I-κBαSR) acts to repress NF-κB activation exclusively in airway epithelial cells, under the transcriptional control of the rat CC10 promoter (CC10-I-κBαSR). Compared with transgene-negative littermates, intranasal instillation of LPS did not induce nuclear translocation of NF-κB in airway epithelium of CC10-I-κBαSR transgenic mice. Consequently, the influx of neutrophils into the airways and secretion of the NF-κB-regulated neutrophilic chemokine, macrophage-inflammatory protein-2, and the inflammatory cytokine, TNF-α, were markedly reduced in CC10-I-κBαSR mice relative to the transgene-negative mice exposed to LPS. Despite an inability to activate NF-κB in airway epithelium, resident alveolar macrophages from transgene-positive mice were capable of activating NF-κB in a manner indistinguishable from transgene-negative mice. These findings demonstrate that airway epithelial cells play a prominent role in orchestrating the airway inflammatory response to LPS and suggest that NF-κB signaling in these cells is important for modulating innate immune responses to microbial products.

Cytokine-induced Activation of Nuclear Factor-κB Is Inhibited by Hydrogen Peroxide through Oxidative Inactivation of IκB Kinase

Rapid activation of the IκB kinase (IKK) complex is considered an obligatory step in the activation of nuclear factor-κB (NF-κB) in response to diverse stimuli. Since oxidants have been implicated in the regulation of NF-κB, the focus of the present study was the activation of IKK by tumor necrosis factor α (TNFα) in the presence or absence of hydrogen peroxide (H2O2). Exposure of mouse alveolar epithelial cells to H2O2 was not sufficient to activate IKK, degrade IκBα, or activate NF-κB. In contrast, TNFα induced IKK activity rapidly and transiently resulting in IκBα degradation and NF-κB activation. Importantly, in the presence of H2O2, the ability of TNFα to induce IKK activity was markedly decreased and resulted in prevention of IκBα degradation and NF-κB activation. Neither tyrosine kinases nor phosphatidylinositol 3-kinases, known regulators of NF-κB by oxidants, were involved in IKK inhibition by H2O2. Direct addition of H2O2 to the immunoprecipitated IKK complex inhibited enzyme activity. Inhibition of IKK activity by H2O2 was associated with direct oxidation of cysteine residues present in the IKK complex and occurred only in enzymatically active IKK. In contrast to previously published observations, our findings demonstrate that the oxidant H2O2 reduces NF-κB activation by inhibiting activated IKK activity.

NF-κB Activation in Airways Modulates Allergic Inflammation but Not Hyperresponsiveness

Airways display robust NF-κB activation and represent targets for anti-inflammatory asthma therapies, but the functional importance of NF-κB activation in airway epithelium remains enigmatic. Therefore, transgenic mice were created in which NF-κB activation is repressed specifically in airways (CC10-IκBαSR mice). In response to inhaled Ag, transgenic mice demonstrated significantly ameliorated inflammation, reduced levels of chemokines, T cell cytokines, mucus cell metaplasia, and circulating IgE compared with littermate controls. Despite these findings, Ag-driven airways hyperresponsiveness was not attenuated in CC10-IκBαSR mice. This study clearly demonstrates that airway epithelial NF-κB activation orchestrates Ag-induced inflammation and subsequent adaptive immune responses, but does not contribute to airways hyperresponsiveness, the cardinal feature that underlies asthma.

Rapid Activation of Nuclear Factor-κB in Airway Epithelium in a Murine Model of Allergic Airway Inflammation

Bronchiolar epithelium is postulated to play a critical role in the orchestration of responses to inhaled allergens, and may contribute to the pathogenesis of asthma. Using a murine model of allergic airway inflammation and hyperresponsiveness, we demonstrate in mice sensitized with ovalbumin (OVA) that following a single challenge with nebulized OVA, a rapid and protracted activation of inhibitor of kappa B kinase (IKK) occurred in lung tissue. IKK activation was followed by nuclear localization of nuclear factor (NF)-kappaB within the bronchiolar epithelium and increased luciferase activity in lungs of mice containing a NF-kappaB-dependent reporter gene. Challenge of sensitized mice with OVA also induced mRNA expression of the chemokines, macrophage inflammatory protein-2 (MIP-2) and eotaxin in lung tissue, which corresponded temporally with the observed influx of neutrophils and eosinophils, respectively, into the airspaces. Using laser capture microdissection and quantitative polymerase chain reaction, we demonstrated that MIP-2 and eotaxin were predominantly expressed in bronchiolar epithelium, in contrast to distal regions of the lungs, which expressed lower or undetectable levels of these mRNAs. These studies strengthen the potential importance of the bronchiolar epithelial cell as a source of production of NF-kappaB-dependent mediators that play a role in asthma.

Asbestos, Lung Cancers, and Mesotheliomas: From Molecular Approaches to Targeting Tumor Survival Pathways

Fifteen years have passed since we published findings in the AJRCMB demonstrating that induction of early response fos/jun proto-oncogenes in rodent tracheal and mesothelial cells correlates with fibrous geometry and pathogenicity of asbestos. Our study was the first to suggest that the aberrant induction of signaling responses by crocidolite asbestos and erionite, a fibrous zeolite mineral associated with the development of malignant mesotheliomas (MMs) in areas of Turkey, led to altered gene expression. New data questioned the widely held belief at that time that the carcinogenic effects of asbestos in the development of lung cancer and MM were due to genotoxic or mutagenic effects. Later studies by our group revealed that proto-oncogene expression and several of the signaling pathways activated by asbestos were redox dependent, explaining why antioxidants and antioxidant enzymes were elevated in lung and pleura after exposure to asbestos and how they alleviated many of the phenotypic and functional effects of asbestos in vitro or after inhalation. Since these original studies, our efforts have expanded to understand the interface between asbestos-induced redox-dependent signal transduction cascades, the relationship between these pathways and cell fate, and the role of asbestos and cell interactions in development of asbestos-associated diseases. Of considerable significance is the fact that the signal transduction pathways activated by asbestos are also important in survival and chemoresistance of MMs and lung cancers. An understanding of the pathogenic features of asbestos fibers and dysregulation of signaling pathways allows strategies for the prevention and therapy of asbestos-related diseases.