The non-provitamin A carotenoid, lutein, inhibits NF-kappaB-dependent gene expression through redox-based regulation of the phosphatidylinositol 3-kinase/PTEN/Akt and NF-kappaB-inducing kinase pathways: role of H(2)O(2) in NF-kappaB activation

Reactive oxygen species (ROS) have been implicated in the regulation of NF-kappaB activation, which plays an important role in inflammation and cell survival. However, the molecular mechanisms of ROS in NF-kappaB activation remain poorly defined. We found that the non-provitamin A carotenoid, lutein, decreased intracellular H(2)O(2) accumulation by scavenging superoxide and H(2)O(2) and the NF-kappaB-regulated inflammatory genes, iNOS, TNF-alpha, IL-1beta, and cyclooxygenase-2, in lipopolysaccharide (LPS)-stimulated macrophages. Lutein inhibited LPS-induced NF-kappaB activation, which highly correlated with its inhibitory effect on LPS-induced IkappaB kinase (IKK) activation, IkappaB degradation, nuclear translocation of NF-kappaB, and binding of NF-kappaB to the kappaB motif of the iNOS promoter. This compound inhibited LPS- and H(2)O(2)-induced increases in phosphatidylinositol 3-kinase (PI3K) activity, PTEN inactivation, NF-kappaB-inducing kinase (NIK), and Akt phosphorylation, which are all upstream of IKK activation, but did not affect the interaction between Toll-like receptor 4 and MyD88 and the activation of mitogen-activated protein kinases. The NADPH oxidase inhibitor apocynin and gp91(phox) deletion reduced the LPS-induced NF-kappaB signaling pathway as lutein did. Moreover, lutein treatment and gp91(phox) deletion decreased the expressional levels of the inflammatory genes in vivo and protected mice from LPS-induced lethality. Our data suggest that H(2)O(2) modulates IKK-dependent NF-kappaB activation by promoting the redox-sensitive activation of the PI3K/PTEN/Akt and NIK/IKK pathways. These findings further provide new insights into the pathophysiological role of intracellular H(2)O(2) in the NF-kappaB signal pathway and inflammatory process.     

Role of extracellular superoxide in neutrophil activation: interactions between xanthine oxidase and TLR4 induce proinflammatory cytokine production

Reactive oxygen species (ROS) contribute to neutrophil activation and the development of acute inflammatory processes in which neutrophils play a central role. However, there is only limited information concerning the mechanisms through which extracellular ROS, and particularly cell membrane-impermeable species, such as superoxide, enhance the proinflammatory properties of neutrophils. To address this issue, neutrophils were exposed to superoxide generating combinations of xanthine oxidase and hypoxanthine or lumazine. Extracellular superoxide generation induced nuclear translocation of nuclear factor-kappaB (NF-kappaB) and increased neutrophil production of the NF-kappaB-dependent cytokines tumor necrosis factor-alpha (TNF-alpha) and macrophage inhibitory protein-2 (MIP-2). In contrast, there were no changes in TNF-alpha or MIP-2 expression when neutrophils lacking Toll-like receptor-4 (TLR4) were exposed to extracellular superoxide. Immunoprecipitation, confocal microscopy, and fluorescence resonance energy transfer (FRET) studies demonstrated association between TLR4 and xanthine oxidase. Exposure of neutrophils to heparin attenuated binding of xanthine oxidase to the cell surface as well as interactions with TLR4. Heparin also decreased xanthine oxidase-induced nuclear translocation of NF-kappaB as well as production of proinflammatory cytokines. These results demonstrate that extracellular superoxide has proinflammatory effects on neutrophils, predominantly acting through an TLR4-dependent mechanism that enhances nuclear translocation of NF-kappaB and increases expression of NF-kappaB-dependent cytokines.     

Thalidomide suppresses NF-kappa B activation induced by TNF and H2O2, but not that activated by ceramide, lipopolysaccharides, or phorbol ester

Thalidomide ([+]-alpha-phthalimidoglutarimide), a psychoactive drug that readily crosses the blood-brain barrier, has been shown to exhibit anti-inflammatory, antiangiogenic, and immunosuppressive properties through a mechanism that is not fully established. Due to the central role of NF-kappaB in these responses, we postulated that thalidomide mediates its effects through suppression of NF-kappaB activation. We investigated the effects of thalidomide on NF-kappaB activation induced by various inflammatory agents in Jurkat cells. The treatment of these cells with thalidomide suppressed TNF-induced NF-kappaB activation, with optimum effect occurring at 50 microg/ml thalidomide. These effects were not restricted to T cells, as other hematopoietic and epithelial cell types were also inhibited. Thalidomide suppressed H(2)O(2)-induced NF-kappaB activation but had no effect on NF-kappaB activation induced by PMA, LPS, okadaic acid, or ceramide, suggesting selectivity in suppression of NF-kappaB. The suppression of TNF-induced NF-kappaB activation by thalidomide correlated with partial inhibition of TNF-induced degradation of an inhibitory subunit of NF-kappaB (IkappaBalpha), abrogation of IkappaBalpha kinase activation, and inhibition of NF-kappaB-dependent reporter gene expression. Thalidomide abolished the NF-kappaB-dependent reporter gene expression activated by overexpression of TNFR1, TNFR-associated factor-2, and NF-kappaB-inducing kinase, but not that activated by the p65 subunit of NF-kappaB. Overall, our results clearly demonstrate that thalidomide suppresses NF-kappaB activation specifically induced by TNF and H(2)O(2) and that this may contribute to its role in suppression of proliferation, inflammation, angiogenesis, and the immune system.     

Co-induction of cell death and survival pathways by phosphoinositide 3-kinase

A single stimulus can induce both the cell death and survival pathway, suggesting that these pathways share common upstream signaling components. In order to define these components, human U937 cells grown in 10% serum were exposed to serum-free media. This treatment resulted in apoptosis, which was found to be mediated by SAPK/JNK. It was previously reported that the serum withdrawal (SW)-induced SAPK activation is mediated by a positive mutual interaction between the reactive oxygen species (ROS) and phosphoinositide 3-kinase (PI3K). This study shows that the ROS/PI3K interaction also induces a NF-kappaB-dependent survival pathway. Despite the role of PI3K, Akt was found to be irrelevant to the activation of SAPK and NF-kappaB. Comparative analyses of SAPK and NF-kappaB for their responses to exogenous H(2)O(2) revealed that SAPK activation requires much higher H(2)O(2) concentrations than those required for NF-kappaB activation. Moreover, high lethal concentrations of H(2)O(2) were found to activate NF-kappaB and SAPK in a PI3K-independent manner. These results suggest that ROS induce both the SAPK-dependent apoptotic and NF-kappaB-mediated survival pathways, and these inducer signals are amplified by PI3K in the SW-triggered pathway. Cell death appears to be favored as this amplification proceeds.     

Microtubule-associated serine/threonine kinase-205 kDa and Fc gamma receptor control IL-12 p40 synthesis and NF-kappa B activation

Stimulation of murine macrophages with LPS results in the coordinated activation of a set of proinflammatory cytokines and costimulatory molecules, including TNF-alpha, IL-6, IL-1, IL-8, IL-12, and CD80. Macrophage LPS-induced synthesis of IL-12 is inhibited following FcgammaR ligation; TNF-alpha secretion is unchanged. We report that microtubule-associated serine/threonine kinase-205 kDa (MAST205) is required for LPS-induced IL-12 synthesis. RNA interference-mediated suppression of MAST205 results in the inhibition of LPS-stimulated IL-12 promoter activity and IL-12 secretion, from both J774 cells and bone marrow-derived macrophages. Similarly, dominant-negative MAST205 mutants inhibit LPS-stimulated IL-12 synthesis and NF-kappaB activation, but do not affect IL-1 or TNF-alpha signaling. Finally, macrophage FcgammaR ligation regulates MAST205 by inducing the rapid ubiquitination and proteasomal degradation of the protein.     

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.     

Antioxidant enzymes suppress nitric oxide production through the inhibition of NF-kappa B activation: role of H(2)O(2) and nitric oxide in inducible nitric oxide synthase expression in macrophages.

Reactive molecules O(-)(2), H(2)O(2), and nitrogen monoxide (NO) are produced from macrophages following exposure to lipopolysaccharide (LPS) and involved in cellular signaling for gene expression. Experiments were carried out to determine whether these molecules regulate inducible nitric oxide synthase (iNOS) gene expression in RAW264.7 macrophages exposed to LPS. NO production was inhibited by the antioxidative enzymes catalase, horseradish peroxidase, and myeloperoxidase but not by superoxide dismutase (SOD). In contrast, the NO-producing activity of LPS-stimulated RAW264.7 cells was enhanced by the NO scavengers hemoglobin (Hb) and myoglobin. The antioxidant enzymes decreased levels of iNOS mRNA and protein in LPS-stimulated RAW264.7 cells, whereas the NOS inhibitor N(G)-monomethyl-L-arginine as well as Hb increased the level of iNOS protein but not mRNA, indicating that NO inhibits iNOS protein expression. NF-kappa B was activated in LPS-stimulated RAW264.7 cells and the activation was significantly inhibited by antioxidant enzymes, but not by Hb. Similar results were obtained using LPS-stimulated rodent peritoneal macrophages. Extracellular O(-)(2) generation by LPS-stimulated macrophages was suppressed by SOD, but not by antioxidative enzymes, while accumulation of intracellular reactive oxygen species was inhibited by antioxidative enzymes, but not by SOD. Exogenous H(2)O(2) induced NF-kappa B activation in macrophages, which was inhibited by catalase and pyrroline dithiocarbamate (PDTC). H(2)O(2) enhanced iNOS expression and NO production in peritoneal macrophages when added with interferon-gamma, and the effect of H(2)O(2) was inhibited by catalase and PDTC. These findings suggest that H(2)O(2) production from LPS-stimulated macrophages participates in the upregulation of iNOS expression via NF-kappa B activation and that NO is a negative feedback inhibitor of iNOS protein expression.     

Hydrogen sulfide inhibits nitric oxide production and nuclear factor-kappaB via heme oxygenase-1 expression in RAW264.7 macrophages stimulated with lipopolysaccharide

Hydrogen sulfide (H(2)S), a regulatory gaseous molecule that is endogenously synthesized by cystathionine gamma-lyase (CSE) and/or cystathionine beta-synthase (CBS) from L-cysteine (L-Cys) metabolism, is a putative vasodilator, and its role in nitric oxide (NO) production is unexplored. Here, we show that at noncytotoxic concentrations, H(2)S was able to inhibit NO production and inducible NO synthase (iNOS) expression via heme oxygenase (HO-1) expression in RAW264.7 macrophages stimulated with lipopolysaccharide (LPS). Both H(2)S solution prepared by bubbling pure H(2)S gas and NaSH, a H(2)S donor, dose dependently induced HO-1 expression through the activation of the extracellular signal-regulated kinase (ERK). Pretreatment with H(2)S or NaHS significantly inhibited LPS-induced iNOS expression and NO production. Moreover, NO production in LPS-stimulated macrophages that are expressing CSE mRNA was significantly reduced by the addition of L-Cys, a substrate for H(2)S, but enhanced by the selective CSE inhibitor beta-cyano-L-alanine but not by the CBS inhibitor aminooxyacetic acid. While either blockage of HO activity by the HO inhibitor, tin protoporphyrin IX, or down-regulation of HO-1 expression by HO-1 small interfering RNA (siRNA) reversed the inhibitory effects of H(2)S on iNOS expression and NO production, HO-1 overexpression produced the same inhibitory effects of H(2)S. In addition, LPS-induced nuclear factor (NF)-kappaB activation was diminished in RAW264.7 macrophages preincubated with H(2)S. Interestingly, the inhibitory effect of H(2)S on NF-kappaB activation was reversed by the transient transfection with HO-1 siRNA, but was mimicked by either HO-1 gene transfection or treatment with carbon monoxide (CO), an end product of HO-1. CO treatment also inhibited LPS-induced NO production and iNOS expression via its inactivation of NF-kappaB. Collectively, our results suggest that H(2)S can inhibit NO production and NF-kappaB activation in LPS-stimulated macrophages through a mechanism that involves the action of HO-1/CO.     

Phosphatase inhibition leads to activation of IkappaB kinase in murine macrophages

We have been interested in elucidating the role of intracellular phosphatase activity in the regulation of immune cell activation. To this end, we treated RAW 264.7 murine macrophages with the phosphatase inhibitor, calyculin-A. Treatment with calyculin-A led to activation of IkappaB kinase, degradation of IkappaBalpha, and induced nuclear translocation and DNA binding of NF-kappaB. Each of these effects occurred in both a time- and dose-dependent manner. In addition, each of these effects was negatively modulated by prior induction of the heat-shock response. Despite clear activation of the IkappaB kinase/IkappaBalpha/NF-kappaB pathway, however, phosphatase inhibition did not lead to increased expression of NF-kappaB-dependent genes. Thus, intracellular phosphatase activity is a central regulator of the NF-kappaB signal transduction pathway and is negatively modulated by heat shock. Inhibition of intracellular phosphatase activity with calyculin-A is not sufficient to induce NF-kappaB-dependent gene expression, demonstrating the complexity of NF-kappaB regulation in immune cells.     

Artemisolide is a typical inhibitor of IkappaB kinase beta targeting cysteine-179 residue and down-regulates NF-kappaB-dependent TNF-alpha expression in LPS-activated macrophages

Nuclear factor (NF)-kappaB regulates a central common signaling for immunity and cell survival. Artemisolide (ATM) was previously isolated as a NF-kappaB inhibitor from a plant of Artemisia asiatica. However, molecular basis of ATM on NF-kappaB activation remains to be defined. Here, we demonstrate that ATM is a typical inhibitor of IkappaB kinase beta (IKKbeta), resulting in inhibition of lipopolysaccharide (LPS)-induced NF-kappaB activation in RAW 264.7 macrophages. ATM inhibited the kinase activity of highly purified IKKbeta and also LPS-induced IKK activity in the cells. Moreover, the effect of ATM on IKKbeta activity was completely abolished by substitution of Cys-179 residue of IKKbeta to Ala residue, indicating direct targeting site of ATM. ATM could inhibit IkappaBalpha phosphorylation in LPS-activated RAW 264.7 cells and subsequently prevent NF-kappaB activation. Further, we demonstrate that ATM down-regulates NF-kappaB-dependent TNF-alpha expression. Taken together, this study provides a pharmacological potential of ATM in NF-kappaB-dependent inflammatory disorders.     

Reactive oxygen species promote TNFalpha-induced death and sustained JNK activation by inhibiting MAP kinase phosphatases

TNFalpha is a pleiotropic cytokine that induces either cell proliferation or cell death. Inhibition of NF-kappaB activation increases susceptibility to TNFalpha-induced death, concurrent with sustained JNK activation, an important contributor to the death response. Sustained JNK activation in NF-kappaB-deficient cells was suggested to depend on reactive oxygen species (ROS), but how ROS affect JNK activation was unclear. We now show that TNFalpha-induced ROS, whose accumulation is suppressed by mitochondrial superoxide dismutase, cause oxidation and inhibition of JNK-inactivating phosphatases by converting their catalytic cysteine to sulfenic acid. This results in sustained JNK activation, which is required for cytochrome c release and caspase 3 cleavage, as well as necrotic cell death. Treatment of cells or experimental animals with an antioxidant prevents H(2)O(2) accumulation, JNK phosphatase oxidation, sustained JNK activity, and both forms of cell death. Antioxidant treatment also prevents TNFalpha-mediated fulminant liver failure without affecting liver regeneration.