Hyperosmotic stress-dependent NFkappaB activation is regulated by reactive oxygen species and IGF-1 in cultured cardiomyocytes

We have recently shown that hyperosmotic stress activates p65/RelB NFkappaB in cultured cardiomyocytes with dichotomic actions on caspase activation and cell death. It remains unexplored how NFkappaB is regulated in cultured rat cardiomyocytes exposed to hyperosmotic stress. We study here: (a) if hyperosmotic stress triggers reactive oxygen species (ROS) generation and in turn whether they regulate NFkappaB and (b) if insulin-like growth factor-1 (IGF-1) modulates ROS production and NFkappaB activation in hyperosmotically-stressed cardiomyocytes. The results showed that hyperosmotic stress generated ROS in cultured cardiac myocytes, in particular the hydroxyl and superoxide species, which were inhibited by N-acetylcysteine (NAC). Hyperosmotic stress-induced NFkappaB activation as determined by IkappaBalpha degradation and NFkappaB DNA binding. NFkappaB activation and procaspase-3 and -9 fragmentation were prevented by NAC and IGF-1. However, this growth factor did not decrease ROS generation induced by hyperosmotic stress, suggesting that its actions over NFkappaB and caspase activation may be due to modulation of events downstream of ROS generation. We conclude that hyperosmotic stress induces ROS, which in turn activates NFkappaB and caspases. IGF-1 prevents NFkappaB activation by a ROS-independent mechanism.     

Suppression of Mycobacterium tuberculosis induced reactive oxygen species (ROS) and TNF-alpha mRNA expression in human monocytes by allicin

Chronic inflammation associated with tumor necrosis factor (TNF)-alpha and reactive oxygen species (ROS) is the hallmark of tuberculosis. Mycobacterium tuberculosis (MTB) directly stimulates human monocytes to secrete TNF-alpha. We show the augmented expression of TNF-alpha mRNA in MTB-infected monocytes by cellular activation and ROS was suppressed by allicin in a dose-dependent manner. Also, allicin enhanced the glutathione peroxidase activity, which correlated inversely with the downregulation of ROS and TNF-alpha in MTB-infected monocytes. Hence, allicin may prove to be a valuable natural antioxidant in combating tuberculosis.     

Spatially distinct production of reactive oxygen species regulates platelet activation

Platelets play a key role in hemostasis and changes in redox balance are known to alter platelet activation and aggregation. Interestingly, activation of platelets leads to production of reactive oxygen species (ROS), but the role(s) of these ROS remain unclear. Using flow cytometry and chemiluminescence, agonist-induced ROS generation was found to be spatially distinct with stimulation through the major collagen receptor GPVI inducing only intraplatelet ROS while thrombin induced production of extracellular ROS. Platelet activation by either the GPVI-selective agonist convulxin or thrombin was differentially regulated by ROS generation. Thus, surface expression of CD62P, CD40L, or activated integrin alphaIIbbeta3 was abrogated by pharmacologic antioxidants but externalization of phosphatidylserine was not inhibited. Furthermore, extracellular antioxidants SOD/catalase markedly inhibited thrombin-, but not convulxin-, induced CD62P expression and alphaIIbbeta3 activation. The data suggest that ROS selectively regulate biochemical steps in platelet activation and that distinct source(s) of ROS and discrete redox-sensitive pathway(s) may control platelet activation in response to GPVI or thrombin stimulation. Thus, targeting ROS with site-specific antioxidants may differentially regulate platelet activation via thrombin or collagen.     

Reactive oxygen species mediate IL-8 expression in Down syndrome candidate region-1-overexpressed cells

Reactive oxygen species (ROS) have been considered to mediate inflammation in Down syndrome (DS). The present study is purposed to examine the mechanism of increased ROS levels and inflammatory cytokine IL-8 expression in Down syndrome candidate region-1 (DSCR1)-transfected cells, by determining ROS levels, IL-8 expression, NF-κB activation, and SOD1 levels in human embryonic kidney (HEK) 293 cells. The cells were treated with an antioxidant N-acetyl cysteine (NAC) or a calcium chelator BAPTA and stimulated with or without IL-1β. As a result, basal levels of ROS, IL-8, and NF-κB-DNA binding activity were higher, and basal SOD1 levels were higher in DSCR1-transfected cells than pcDNA-transfected cells. BAPTA and NAC inhibited increase in ROS (intracellular and mitochondrial levels) in DSCR-1-transfected cells without treatment of IL-1β. DSCR1 transfection-induced changes were increased by treatment with IL-1β, which was suppressed by NAC and BAPTA. Transfection of SOD1 inhibited ROS levels in DSCR1-transfected cells. In conclusion, ROS activate NF-κB and IL-8 induction in DSCR1-transfected cells in a calcium-dependent manner, which is augmented by IL-1β since IL-1β increases calcium and ROS levels in the cells. Reducing ROS levels by treatment of antioxidants may be beneficial for preventing DS-associated inflammation by suppressing cytokine expression.     

IL-1beta induces MMP-9 via reactive oxygen species and NF-kappaB in murine macrophage RAW 264.7 cells

IL-1beta increased the production of proenzyme of MMP-9 (pro-MMP-9) in a time- and dose-dependent manner in murine macrophage RAW 264.7 cells. However, the production of MMP-2 was not significantly changed by IL-1beta treatment. The intracellular H(2)O(2) content, as determined with H(2)O(2)-sensitive probe 2('),7(')-dichlorodihydrofluorescein, also increased after IL-1beta treatment (5ng/ml). In addition, exogenous H(2)O(2) (50 microM) was found to increase the production of pro-MMP-9. Transient transfection study using a MMP-9 promoter-reporter construct showed that IL-1beta enhanced the MMP-9 promoter activity. Electrophoretic mobility shift assay and site-directed mutagenesis study on the consensus binding site for NF-kappaB revealed that the activation of NF-kappaB is required for the IL-1beta-induced activation of MMP-9 promoter. N-acetylcysteine, an antioxidant, could abrogate the production of pro-MMP-9, H(2)O(2) generation, and activation of NF-kappaB and MMP-9 promoter. These results suggest that IL-1beta upregulates the MMP-9 expression via production of reactive oxygen species and activation of NF-kappaB in RAW 264.7 cells.     

Importance of lipid rafts for lysophosphatidylcholine-induced caspase-1 activation and reactive oxygen species generation

Lipid rafts play an important role in regulating cellular processes and functions. Here, we demonstrate that in microglia stimulated with the pro-inflammatory lipid lysophosphatidylcholine (LPC), caspase-1 activation and NADPH oxidase activity depend on intact lipid rafts. Disruption of lipid rafts with methyl-β-cyclodextrin, fumonisin B1 or nystatin prevented LPC-stimulated caspase-1 activation and reactive oxygen species (ROS) production, whereas LPC-induced Na⁺ influx remained unaffected. Since ROS regulate caspase-1 activity in LPC-stimulated microglia, the effects of lipid raft-disrupting agents on caspase-1 activation can be related to their inhibition of NADPH oxidase-mediated ROS production.     

DJ-1 is an indicator for endogenous reactive oxygen species elicited by endotoxin

We previously found that DJ-1 protein of pI 5.8 (DJ-1/5.8) increased on 2D gels as DJ-1 of pI 6.2 (DJ-1/6.2) decreased, upon exposure of human cells to sublethal levels of oxidative stress, such as H₂O₂ and paraquat. Here, we show that the DJ-1/5.8 increases concomitantly with endogenous production of reactive oxygen species (ROS) under endotoxin-induced inflammatory conditions. Lipopolysaccharide (LPS) significantly increased the expression of DJ-1/5.8 in murine peritoneal macrophages (MΦ) and a murine macrophage cell line (J774). Diphenylene iodonium, a flavoenzyme inhibitor, blocked the effect of LPS on DJ-1/5.8 expression. Aminoguanidine (AG), a selective inhibitor of type II nitric oxide synthase, had no effect on the DJ-1/5.8 expression, but suppressed accumulation of nitrite in the culture medium after LPS treatment. We also examined the expression of DJ-1/5.8 in lung, since acute lung injury is seen in endotoxin shock. When female mice (6-weeks old) were intraperitoneally given LPS (10 mg/kg), myeloperoxidase (MPO) activity in lung, a marker of neutrophil infiltration, was transiently raised by 3.5 fold. The expression of DJ-1/5.8 in lung was enhanced and then reverted to the control level, in parallel with the MPO activity. These results, taken together, suggest that the DJ-1/5.8 might increase in response to endogenously produced ROS, probably due to activation of NADPH oxidase, and imply that DJ-1 may be useful as an endogenous indicator of oxidative stress status in vivo.     

The role of UCP 1 in production of reactive oxygen species by mitochondria isolated from brown adipose tissue

We provide evidence that ablation or inhibition of, uncoupling protein 1 increases the rate of reactive oxygen containing species production by mitochondria from brown adipose tissue, no matter what electron transport chain substrate is used (succinate, glycerol-3-phosphate or pyruvate/malate). Consistent with these data are our observations that (a) the mitochondrial membrane potential is maximal when uncoupling protein 1 is ablated or inhibited and (b) oxygen consumption rates in mitochondria from uncoupling protein 1 knock-out mice, are significantly lower than those from wild-type mice, but equivalent to those from wild-type mice in the presence of GDP. In summary, we show that uncoupling protein 1 can affect reactive oxygen containing species production by isolated mitochondria from brown adipose tissue.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria

Huntington’s disease results from expansion of the polyglutamine (PolyQ) domain in the huntingtin protein. Although the cellular mechanism by which pathologic-length PolyQ protein causes neurodegeneration is unclear, mitochondria appear central in pathogenesis. We demonstrate in isolated mitochondria that pathologic-length PolyQ protein directly inhibits ADP-dependent (state 3) mitochondrial respiration. Inhibition of mitochondrial respiration by PolyQ protein is not due to reduction in the activities of electron transport chain complexes, mitochondrial ATP synthase, or the adenine nucleotide translocase. We show that pathologic-length PolyQ protein increases the production of reactive oxygen species in isolated mitochondria. Impairment of state 3 mitochondrial respiration by PolyQ protein is reversed by addition of the antioxidants N-acetyl-l-cysteine or cytochrome c. We propose a model in which pathologic-length PolyQ protein directly inhibits mitochondrial function by inducing oxidative stress.     

The effect of reactive oxygen species on ethylene production induced by osmotic stress in etiolated mungbean seedling

After 10 h osmotic stress in 25% polyethylene glycol (PEG6000) solution (–1.8 MPa) at 25 °C in darkness, the etiolated mungbean seedlings were transferred to pure water for recovery. The ethylene release rate and the level of reactive oxygen species (ROS), including superoxide radical (O₂^–) and hydrogen peroxide (H₂O₂), were investigated during the recovery process. The results showed that ethylene production rate and amount of ROS increased dramatically after osmotic stress, and a close correlation was observed between ethylene release rate and concentrations of ROS. Inhibitors of ethylene biosynthesis, aminoethoxyvinylglycine (AVG) or aminooxyacetic acid (AOA), could reduce the ethylene release rate, but had no significant influence to the content of O₂^– and H₂O₂. As well as, silver thiosulfate (STS), an inhibitor of ethylene action, exhibited no obvious effect to the concentration of ROS, showing stress-inducible ethylene was not the cause for the increase of stress-inducible ROS. On the other hand, exogenous generator of superoxide radical (methylviologen, MV, or sodium dithionite, Na₂S₂O₄) could enhance the ethylene production evidently, which could be inhibited by exogenous scavenger of superoxide radical (superoxide dismutase, SOD, or 1, 4-diazabicyclo (2,2,2) octane, DABCO). However, either exogenous H₂O₂ or catalase (CAT) had no significant influence on ethylene production. The results suggested that it was superoxide radical but not H₂O₂which was involved directly in osmotic stress-inducible ethylene biosynthesis. The dual-role of superoxide radical on stress ethylene biosynthesis was also discussed.     

Calcium-induced generation of reactive oxygen species in brain mitochondria is mediated by permeability transition

Mitochondrial uptake of calcium in excitotoxicity is associated with subsequent increase in reactive oxygen species (ROS) generation and delayed cellular calcium deregulation in ischemic and neurodegenerative insults. The mechanisms linking mitochondrial calcium uptake and ROS production remain unknown but activation of the mitochondrial permeability transition (mPT) may be one such mechanism. In the present study, calcium increased ROS generation in isolated rodent brain and human liver mitochondria undergoing mPT despite an associated loss of membrane potential, NADH and respiration. Unspecific permeabilization of the inner mitochondrial membrane by alamethicin likewise increased ROS independently of calcium, and the ROS increase was further potentiated if NAD(H) was added to the system. Importantly, calcium per se did not induce a ROS increase unless mPT was triggered. Twenty-one cyclosporin A analogs were evaluated for inhibition of calcium-induced ROS and their efficacy clearly paralleled their potency of inhibiting mPT-mediated mitochondrial swelling. We conclude that while intact respiring mitochondria possess powerful antioxidant capability, mPT induces a dysregulated oxidative state with loss of GSH- and NADPH-dependent ROS detoxification. We propose that mPT is a significant cause of pathological ROS generation in excitotoxic cell death.     

Role of reactive oxygen species in LPS-induced production of prostaglandin E2 in microglia

We determined the roles of reactive oxygen species (ROS) in the expression of cyclooxygenase-2 (COX-2) and the production of prostaglandin E2 (PGE2) in lipopolysaccharide (LPS)-activated microglia. LPS treatment increased intracellular ROS in rat microglia dose-dependently. Pre-treatment with superoxide dismutase (SOD)/catalase, or SOD/catalase mimetics that can scavenge intracellular ROS, significantly attenuated LPS-induced release in PGE2. Diphenylene iodonium (DPI), a non-specific NADPH oxidase inhibitor, decreased LPS-induced PGE2 production. In addition, microglia from NADPH oxidase-deficient mice produced less PGE2 than those from wild-type mice following LPS treatment. Furthermore, LPS-stimulated expression of COX-2 (determined by RT-PCR analysis of COX-2 mRNA and western blot for its protein) was significantly reduced by pre-treatment with SOD/catalase or SOD/catalase mimetics. SOD/catalase mimetics were more potent than SOD/catalase in reducing COX-2 expression and PGE2 production. As a comparison, scavenging ROS had no effect on LPS-induced nitric oxide production in microglia. These results suggest that ROS play a regulatory role in the expression of COX-2 and the subsequent production of PGE2 during the activation process of microglia. Thus, inhibiting NADPH oxidase activity and subsequent ROS generation in microglia can reduce COX-2 expression and PGE2 production. These findings suggest a potential therapeutic intervention strategy for the treatment of inflammation-mediated neurodegenerative diseases.     

Mountain cedar pollen induces IgE-independent mast cell degranulation, IL-4 production, and intracellular reactive oxygen species generation

Cedar pollens cause severe allergic disease throughout the world. We have previously characterized allergenic pollen glycoproteins from mountain cedar (Juniperus ashei) that bind to allergen-specific immunoglobulin E (IgE). In the present report, we investigated an alternative pathway of mast cell activation by mountain cedar pollen extract through IgE-independent mechanisms. We show that mountain cedar pollen directly induces mast cell serotonin and IL-4 release and enhances release induced by IgE cross-linking. Concomitant with mediator release, high levels of intracellular reactive oxygen species (ROS) were generated, and both ROS and serotonin release were inhibited by anti-oxidants. These findings suggest that alternative mechanisms exist whereby pollen exposure enhances allergic inflammatory mediator release through mechanisms that involve ROS. These mechanisms have the potential for enhancing the allergenic potency of pollens.     

NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species production

NOD-like receptors (NLRs) are a family of intracellular sensors of microbial- or danger-associated molecular patterns. Here, we report the identification of NLRX1, which is a new member of the NLR family that localizes to the mitochondria. NLRX1 alone failed to trigger most of the common signalling pathways, including nuclear factor-kappaB (NF)-kappaB- and type I interferon-dependent cascades, but could potently trigger the generation of reactive oxygen species (ROS). Importantly, NLRX1 synergistically potentiated ROS production induced by tumour necrosis factor alpha, Shigella infection and double-stranded RNA, resulting in amplified NF-kappaB-dependent and JUN amino-terminal kinases-dependent signalling. Together, these results identify NLRX1 as a NLR that contributes to the link between ROS generation at the mitochondria and innate immune responses.     

Apigenin-induced apoptosis is mediated by reactive oxygen species and activation of ERK1/2 in rheumatoid fibroblast-like synoviocytes

Fibroblast-like synovial cells play a crucial role in the pathophysiology of rheumatoid arthritis (RA), as these cells are involved in inflammation and joint destruction. Apigenin, a dietary plant-flavonoid, is known to have many functions in animal cells including anti-proliferative and anticancer activities, but its role in human rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) has not been reported. In this study, we investigated the roles of apigenin in RA-FLSs. The survival rate decreased, and apoptotic cell death was induced by apigenin treatment in RA-FLSs. Apigenin treatment resulted in activation of the mitogen-activated protein kinase (MAPK) ERK1/2, and pretreatment with an ERK inhibitor PD98059 dramatically reduced apigenin-induced apoptosis. We found that apigenin-mediated production of a large amount of intracellular reactive oxygen species (ROS) caused activation of ERK1/2 and apoptosis; treatment with the antioxidant Tiron strongly inhibited the apigenin-induced generation of ROS, phosphorylation of ERK1/2, and apoptotic cell death. Apigenin-induced apoptotic cell death was mediated through activation of the effectors caspase-3 and caspase-7, and was blocked by pretreatment with Z-VAD-FMK (a pan-caspase inhibitor). These results showed that apigenin-induced ROS and oxidative stress-activated ERK1/2 caused apoptotic cell death in apigenin-treated RA-FLSs.