We report a novel function of peroxiredoxin-1 (Prx-1) in the ASK1-mediated signaling pathway. Prx-1 interacts with ASK1 via the thioredoxin-binding domain of ASK1 and this interaction is highly inducible by H 2 O 2 . However, catalytic mutants of Prx1, C52A, C173A, and C52A/C173A, could not undergo H 2 O 2 inducible interactions, indicating that the redoxsensitive catalytic activity of Prx-1 is required for the interaction with ASK1. Prx-1 overexpression inhibited the activation of ASK1, and resulted in the inhibition of downstream signaling cascades such as the MKK3/6 and p38 pathway. In Prx-1 knockdown cells, ASK1, p38, and JNK were quickly activated, leading to apoptosis in response to H 2 O 2 . These findings suggest a negative role of Prx-1 in ASK1-induced apoptosis.

Peroxiredoxin 6 (Prdx6) is a bifunctional enzyme with peroxidase activity and Ca 2؉ -independent phospholipase A2 (iPLA2) activity. Here, we report that H 2 O 2 -induced cellular toxicity acts through Prdx6 hyperoxidation. Under high concentrations of H 2 O 2 (>100 M), Prdx6, and 2-Cys Prdxs were hyperoxidized. Contrary to hyperoxidation of 2-Cys Prdxs, hyperoxidation of Prdx6 was irreversible in vivo. Surprisingly, H 2 O 2 -induced cell cycle arrest at the G2/M transition correlated with hyperoxidation and increased iPLA2 activity of Prdx6. This arrest was also associated with up-regulation of p53 and p21 and with downregulation of cyclin B1. Furthermore, the H 2 O 2 -mediated increase in iPLA2 activity was dramatically abolished in a hyperoxidation mutant (C47A), an iPLA2 mutant (S32A), and a double mutant (C47A/S32A) of Prdx6, demonstrating the essential requirement of Prdx6 C47 hyperoxidation for its iPLA2 activity. Together, our results demonstrate that H 2 O 2 -mediated hyperoxidation of Prdx6 induces cell cycle arrest at the G2/M transition through up-regulation of iPLA2 activity. Peroxiredoxins (Prdxs)2 are a family of peroxidases that reduce mainly hydrogen peroxide (H 2 O 2 ) and alkyl hydroperoxides to water and alcohol, respectively (1, 2). Prdxs are classified as either 1-Cys or 2-Cys Prdxs, based on whether the protein contains one or two conserved cysteine residues, respectively. In mammals, six members of the Prdx family have been described. Five of these (Prdx1, Prdx2, Prdx3, Prdx4, and Prdx5) are 2-Cys enzymes that use thioredoxin as the electron donor of their catalytic cycle (3, 4). In contrast, Prdx6, the sole mammalian 1-Cys Prdx, does not use thioredoxin as a reductant.In addition to peroxidase activity, Prdx6 has a Ca 2ϩ -independent phospholipase A2 (iPLA2) activity (5). The peroxidase activity of Prdx6 has been widely studied in cells and animal models for its antioxidant properties that provide protection against the harmful consequence of oxidative stress (6 -8). However, the iPLA2 activity of Prdx6 remains poorly understood. Considering the many functions of iPLA2 activity, including cell cycle progression, apoptosis, and tumorigenesis (9), it may also play an important role in either H 2 O 2 -mediated signaling or H 2 O 2 -related cellular events.Prdxs like Prdx6 use cysteine as a catalytic center, rather than selenocysteine which characterizes the glutathione peroxidases (GPx) (2, 3). The N-terminal conserved cysteines (Cys 51 of Prdx1) of Prdx1-5 are selectively oxidized by H 2 O 2 to Cys-SOH (10). The unstable Cys 51 -SOH reacts with Cys 172-SH of another Prdx molecule, creating a homodimer through an intermolecular disulfide bond. The disulfide is then reduced back to the Prdx active thiol form by the thioredoxin-thioredoxin reductase system (11-16). Under oxidative stress condition, however, the sulfenic intermediate is susceptible to hyperoxidation and thus occasionally hyperoxidized by H 2 O 2 , leading to the formation of sulfinic acid (Cys-SO 2 H) or sulfonic acid (Cys-SO 3...

Oxidative stress is implicated in the pathogenesis of allergic asthma and remains an attractive target for the prevention of the disease. Herein, we investigated the anti-inflammatory effects of apocynin, a NADPH oxidase (NOX) inhibitor, in both in vitro and in vivo allergen-induced experimental asthma mediated by Th2 hyperresponsiveness. Apocynin showed potential antioxidant activities and inhibitory effects on the activation of redox-sensitive transcription factors, such as NF-jB and AP-1, induced by pro-inflammatory stimuli, such as TNF-a, lipopolysaccharide and Poly I:C, and that inhibited the production of pro-inflammatory cytokines, such as TNF-a, IL-1b and IL-6. In in vivo experimental asthma model, moreover, apocynin significantly attenuated ovalbumin-induced airway hyperresponsiveness and inflammation, as shown by the attenuation of total inflammatory cell and soluble product influx into bronchoalveolar lavage fluid, such as macrophages, eosinophils, IL-4, IL-5, IL-12, IL-13 and TNF-a. Apocynin also significantly reduced lung inflammation in the tissues. Altogether, these results suggest that apocynin may be useful in the treatment of inflammatory diseases induced by oxidative stress through NOX activity. Keywords: apocynin; NADPH oxidase; oxidative stress; asthma; inflammation; NF-kB Asthma is a chronic inflammatory lung disease characterized by infiltration of inflammatory cells, including eosinophils, and airway hyperresponsiveness (AHR). 1-5 T-helper type 2 (Th2) cells, together with other inflammatory cells such as macrophages, eosinophils, mast cells and B cells, have critical roles in the initiation, development and chronicity of this disease. 1 Upon challenge with various allergens, these inflammatory cells infiltrate into the airway and contribute to the production of Th2 cytokines, such as IL-4, IL-5 and IL-13, which are found at elevated levels in asthmatic lungs. 1-5 Th2 cytokines are pivotal for B cell maturation, IgR synthesis, airway eosinophilia, mucus secretion and ultimately AHR. Specifically, IL-4 regulates allergic inflammation by promoting Th2 cell differentiation, controlling the production of IgE in B cells, stimulating B cell proliferation, inducing the upregulation of MHC class II molecules and increasing the expression of an inducible form of the low-affinity receptor for IgE (FcRII or CD23) on B lymphocytes and macrophages. 4,5 IL-5 influences the production, maturation and activation of eosinophils. 4,5 IL-13 is a potent modulator of human monocyte and B cell function. 5 IL-13 is also capable of inducing the expression of CD23 on purified human B cells and acts as a switch factor directing IgE synthesis. 5 Increased reactive oxygen species (ROS) generation, which results in imbalance between oxidative forces and the antioxidant defense systems, has been implicated in the pathogenesis of asthma. 6-8 ROS are capable of eliciting a variety of pathological changes, including the peroxidation of lipids, proteins and DNA, and the generation of chemo-attractants, enhancement of AHR,...

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Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-α, IL-1β, and LPS resulted in significant ROS production and NF-κB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-α levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b + macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.

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dTransforming growth factor ␤ (TGF-␤)-activated kinase 1 (TAK1) is a key regulator in the signals transduced by proinflammatory cytokines and Toll-like receptors (TLRs). The regulatory mechanism of TAK1 in response to various tissue types and stimuli remains incompletely understood. Here, we show that ribosomal S6 kinase 1 (S6K1) negatively regulates TLR-mediated signals by inhibiting TAK1 activity. S6K1 overexpression causes a marked reduction in NF-B and AP-1 activity induced by stimulation of TLR2 or TLR4. In contrast, S6K1؊/؊ and S6K1 knockdown cells display enhanced production of inflammatory cytokines. Moreover, S6K1؊/؊ mice exhibit decreased survival in response to challenge with lipopolysaccharide (LPS). We found that S6K1 inhibits TAK1 kinase activity by interfering with the interaction between TAK1 and TAB1, which is a key regulator protein for TAK1 catalytic function. Upon stimulation with TLR ligands, S6K1 deficiency causes a marked increase in TAK1 kinase activity that in turn induces a substantial enhancement of NF-B-dependent gene expression, indicating that S6K1 is negatively involved in the TLR signaling pathway by the inhibition of TAK1 activity. Our findings contribute to understanding the molecular pathogenesis of the impaired immune responses seen in type 2 diabetes, where S6K1 plays a key role both in driving insulin resistance and modulating TLR signaling.T ransforming growth factor ␤-activated kinase 1 (TAK1) is a member of the mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) family (1). TAK1 is essential for the production of tumor necrosis factor (TNF-␣) and other inflammatory mediators by activating several MAPKs, such as p38␣ MAPK, Jun N-terminal protein kinases 1 and 2 (JNK1 and JNK2), and extracellular signal-regulated kinases 1 and 2 (ERK1/2). TAK1 also plays a key regulatory role in several cytokine-mediated innate immunity signal transduction cascades, including interleukin-1 (IL-1) and the downstream signaling of Toll-like receptors (TLRs) and NOD1/2 (2, 3). In these pathways, various proinflammatory cytokines and TLR agonists trigger TAK1 activity, leading to its autophosphorylation and subsequent recruitment to the IB kinase (IKK) complex, ultimately resulting in the activation of the transcription factor NF-B and the upregulation of genes encoding proinflammatory cytokines, chemokines, adhesion molecules, and proteolytic enzymes.Several binding partners of TAK1, including TAK1-binding protein 1 (TAB1), TAB2, and TAB3, have been implicated in the regulation of TAK1 activity in response to various stimuli (1, 4). Previous reports demonstrated that the native forms of TAK1 comprise a catalytic kinase subunit in complex with the regulatory subunit TAB1 and either of two homologous proteins, TAB2 and TAB3 (2, 5, 6). Importantly, it has been reported that TAB1 might play a key role in the regulation of the TAK1 complex (7,8). Studies with TAB1-deficient mouse embryonic fibroblasts (TAB1 Ϫ/Ϫ MEFs) demonstrated that TAB1 is able to recruit p38␣ MAPK to the TAK1 complex f...

Salt-inducible kinases (SIKs) are a family of related serine-threonine kinases and are involved in controlling various metabolisms such as liver glucose homeostasis, hepatic lipogenesis, steroidogenesis, and adipogenesis. Here we investigated the regulatory role of SIK proteins in Toll-like receptor 4 (TLR4)-mediated signaling. Overexpression of SIK1 and SIK3, but not SIK2, significantly inhibited nuclear factor-κB activity in response to lipopolysaccharide stimulation and affected the expression of proinflammatory cytokines. In contrast, both SIK1(KD) and SIK3(KD) Raw 264.7 cells exhibit dramatic elevations of nuclear factor-κB activation and activations of downstream signaling molecules, such as TGF-β-activated kinase 1, p38, and c-Jun N-terminal kinase, in response to TLR4 stimulation, indicating that SIK1 and SIK3 are negatively involved in the TLR4-mediated signaling. Through biochemical studies, we found that SIK1 and SIK3 interact with TGF-β-activated kinase 1-binding protein 2 (TAB2), and interrupt the functional complex of TAB2-TNF receptor-associated factor 6 (TRAF6). Interestingly, the molecular interruption is induced to suppress the ubiquitination of TRAF6 in response to TLR4 stimulation. These result suggest that SIK1 and SIK3 negatively regulate TLR4-mediated signaling through the interruption of TAB2-TRAF6 complex and thereby the inhibition of ubiquitination of TRAF6. The present findings can be useful for a better understanding of multilevel interactions between the metabolic and immune systems.