Mitochondria-derived Hydrogen Peroxide Selectively Enhances T Cell Receptor-initiated Signal Transduction

T cell receptor (TCR)-initiated signal transduction is reported to increase production of intracellular reactive oxygen species, such as superoxide (O₂^⨪) and hydrogen peroxide (H₂O₂), as second messengers. Although H₂O₂ can modulate signal transduction by inactivating protein phosphatases, the mechanism and the subcellular localization of intracellular H₂O₂ as a second messenger of the TCR are not known. The antioxidant enzyme superoxide dismutase (SOD) catalyzes the dismutation of highly reactive O₂^⨪ into H₂O₂ and thus acts as an intracellular generator of H₂O₂. As charged O₂^⨪ is unable to diffuse through intracellular membranes, cells express distinct SOD isoforms in the cytosol (Cu,Zn-SOD) and mitochondria (Mn-SOD), where they locally scavenge O₂^⨪ leading to production of H₂O₂. A 2-fold organelle-specific overexpression of either SOD in Jurkat T cell lines increases intracellular production of H₂O₂ but does not alter the levels of intracellular H₂O₂ scavenging enzymes such as catalase, membrane-bound peroxiredoxin1 (Prx1), and cytosolic Prx2. We report that overexpression of Mn-SOD enhances tyrosine phosphorylation of TCR-associated membrane proximal signal transduction molecules Lck, LAT, ZAP70, PLCγ1, and SLP76 within 1 min of TCR cross-linking. This increase in mitochondrial H₂O₂ specifically modulates MAPK signaling through the JNK/cJun pathway, whereas overexpressing Cu,Zn-SOD had no effect on any of these TCR-mediated signaling molecules. As mitochondria translocate to the immunological synapse during TCR activation, we hypothesize this translocation provides the effective concentration of H₂O₂ required to selectively modulate downstream signal transduction pathways.     

Regulation of reactive oxygen species generation in cell signaling

Reactive oxygen species (ROS) including superoxide anion and hydrogen peroxide (H₂O₂) are thought to be byproducts of aerobic respiration with damaging effects on DNA, protein, and lipid. A growing body of evidence indicates, however, that ROS are involved in the maintenance of redox homeostasis and various cellular signaling pathways. ROS are generated from diverse sources including mitochondrial respiratory chain, enzymatic activation of cytochrome p450, and NADPH oxidases further suggesting involvement in a complex array of cellular processes. This review summarizes the production and function of ROS. In particular, how cytosolic and membrane proteins regulate ROS generation for intracellular redox signaling will be detailed.     

Iso-superoxide dismutase in <Emphasis Type="Italic">Deinococcus grandis</Emphasis>, a UV resistant bacterium

Deinococcus grandis possesses two types of superoxide dismutase (SOD, E. C. 1.15.1.1.) that show distinct electrophoretic behavior, one that migrates slowly and the other that migrates rapidly (SOD-1 and SOD-2, respectively). In this study, SOD-1 was uniformly and abundantly detected, regardless of growth phase, whereas SOD-2 was not detected during early growth, but was detectable from the exponential growth phase. In addition, a substantial increase in SOD-2 was observed in cells that were treated with potassium superoxide or UV, which suggests that SOD-2 is an inducible protein produced in response to stressful environments. Insensitivity of SOD-1 to both H₂O₂ and cyanide treatment suggests that SOD-1 is MnSOD. However, SOD-2 would be FeSOD, since it lost activity in response to H₂O₂ treatment, but not to cyanide. Localization studies of D. grandis iso-SODs in sucrose-shocked cells suggest that SOD-1 is a membrane-associated enzyme, whereas SOD-2 is a cytosolic enzyme. In conclusion, SOD-1 seems to be an essential constitutive enzyme for viability and SOD-2 appears to be an inducible enzyme that is probably critical for survival upon UV irradiation and oxidative stress.     

Salicylic acid-induced superoxide generation catalyzed by plant peroxidase in hydrogen peroxide-independent manner

It has been reported that salicylic acid (SA) induces both immediate spike and long lasting phases of oxidative burst represented by the generation of reactive oxygen species (ROS) such as superoxide anion radical (O₂^•−). In general, in the earlier phase of oxidative burst, apoplastic peroxidase are likely involved and in the late phase of the oxidative burst, NADPH oxidase is likely involved. Key signaling events connecting the 2 phases of oxidative burst are calcium channel activation and protein phosphorylation events. To date, the known earliest signaling event in response to exogenously added SA is the cell wall peroxidase-catalyzed generation of O₂^•− in a hydrogen peroxide (H₂O₂)-dependent manner. However, this model is incomplete since the source of the initially required H₂O₂ could not be explained. Based on the recently proposed role for H₂O₂-independent mechanism for ROS production catalyzed by plant peroxidases (Kimura et al., 2014, Frontiers in Plant Science), we hereby propose a novel model for plant peroxidase-catalyzed oxidative burst fueled by SA.     

Effects of hydrogen peroxide (H2O2) on alkaline phosphatase activity and matrix mineralization of odontoblast and osteoblast cell lines

Hydrogen peroxide (H₂O₂), an oxidizing agent, has been widely used as a disinfectant. Recently, because of its reactive properties, H₂O₂ has also been used as a tooth bleaching agent in dental care. This is a cause for concern because of adverse biological effects on the soft and hard tissues of the oral environment. To investigate the influence of H₂O₂ on odontoblasts, the cells producing dentin in the pulp, we assessed cellular viability, generation of reactive oxygen species (ROS), alkaline phosphatase (ALP) activity, and nodule formation of an odontoblastic cell line (MDPC-23) after treatment with H₂O₂, and compared those with the effects on preosteoblastic MC3T3-E1 cells. Cytotoxic effects of H₂O₂ began to appear at 0.3 mmol/L in both MDPC-23 and MC3T3-E1 cells. At that concentration, the accumulation of intracellular ROS was confirmed by a fluorescent probe, DCFH-DA. Although more ROS were detected in MDPC-23, the increasing pattern and rate are similar between the two cells. When the cells were treated with H₂O₂ at concentrations below 0.3 mmol/L, MDPC-23 displayed a significant increase in ALP activity and mineralized bone matrix, while MC3T3-E1 cells showed adverse effects of H₂O₂. It is known that ROS are generally harmful by-products of aerobic life and represent the primary cause of aging and numerous diseases. These data, however, suggest that ROS can induce in vitro cell differentiation, and that they play a more complex role in cell physiology than simply causing oxidative damage.     

Index

Hydrogen peroxide (H₂O₂) can induce cell damage by generating reactive oxygen species (ROS), resulting in DNA damage and cell death. The aim of this study is to elucidate the protective effects of fisetin (3,7,3′,4′,-tetrahydroxy flavone) against H₂O₂-induced cell damage. Fisetin reduced the level of superoxide anion, hydroxyl radical in cell free system, and intracellular ROS generated by H₂O₂. Moreover, fisetin protected against H₂O₂-induced membrane lipid peroxidation, cellular DNA damage, and protein carbonylation, which are the primary cellular outcomes of H₂O₂ treatment. Furthermore, fisetin increased the level of reduced glutathione (GSH) and expression of glutamate-cysteine ligase catalytic subunit, which is decreased by H₂O₂. Conversely, a GSH inhibitor abolished the cytoprotective effect of fisetin against H₂O₂-induced cells damage. Taken together, our results suggest that fisetin protects against H₂O₂-induced cell damage by inhibiting ROS generation, thereby maintaining the protective role of the cellular GSH system.     

Knock-out of metacaspase and/or cytochrome c results in the activation of a ROS-independent acetic acid-induced programmed cell death pathway in yeast

To gain further insight into yeast acetic acid-induced programmed cell death (AA-PCD) we analyzed the effects of the antioxidant N-acetyl-l-cysteine (NAC) on cell viability, hydrogen peroxide (H₂O₂) production, DNA fragmentation, cytochrome c (cyt c) release and caspase-like activation in wild type (wt) and metacaspase and/or cyt c-lacking cells. We found that NAC prevents AA-PCD in wt cells, by scavenging H₂O₂ and by inhibiting both cyt c release and caspase-like activation. This shows the occurrence of a reactive oxygen species (ROS)-dependent AA-PCD. Contrarily no NAC dependent change in AA-PCD of mutant cells was detectable, showing that a ROS-independent AA-PCD can also occur.     

The production and scavenging of reactive oxygen species in the plastoquinone pool of chloroplast thylakoid membranes

Reactive oxygen species (ROS) resulting from oxygen reduction, superoxide anion radical O and hydrogen peroxide H ₂O₂ are very significant in the cell metabolism of aerobic organisms. They can be destructive and lead to apoptosis and they can also serve as signal molecules. In the light, chloroplasts are known to be one of the main sources of ROS in plants. However, the components involved in oxygen reduction and the detailed chemical mechanism are not yet well established. The present review describes the experimental data and theoretical considerations that implicate the plastoquinone pool (PQ‐pool) in this process. The evidence indicates that the PQ‐pool has a dual role: (1) the reduction of O₂ by plastosemiquinone to superoxide and (2) the reduction of superoxide by plastohydroquinone to hydrogen peroxide. The second role represents not only the scavenging of superoxide, but also the generation of hydrogen peroxide as an important signaling molecule. The regulatory and protective functions of the PQ‐pool are discussed in the context of these reactions.     

Synthesis of Isozymes of Superoxide Dismutase in Maize Leaves in Response to O3, SO2 and Elevated O2

Matters, G. L. and Scandalios, J. G. 1987. Synthesis of isozymesof superoxide dismutase in maize leaves in response to O₃ SO₂and elevated O₂.—J. exp. Bot 38: 842–852. The activities of the enzymes superoxide dismutase (SOD) andcatalase were determined in maize leaves treated with O₃or SO₂for8 h, or with elevated levels of oxygen for up to 96 h. NeitherO₃nor SO₂significantly increased the levels of superoxide dismutaseor catalase activity. However, after 72 h in an atmosphere containing90% oxygen, superoxide dismutase activity was increased, butnot the activities of catalase, ascorbate pcroxidase, and malatedehydrogenase. Immunological analysis showed that amounts ofthe cytosolic superoxide dismutase isozymes, SOD-2 and SOD-4,were increased by the elevated oxygen but not the chroloplast(SOD-1) or mitochondrial (SOD-3) isozymes. Immunoprecipitationof translation products of leaf polysomes indicated that thehigher levels of SOD-2 and SOD-4 were due to increased amountsof polysome-bound mRNA coding for these proteins. The specificresponse of SOD-2 and SOD-4 to 90% oxygen treatments contrastswith the increase in all SOD isozymes in maize leaves treatedwith the herbicide paraquat. Key words: Air pollutants, maize, oxidative stress, oxygen, superoxide dismutase     

Grain and Bean Lysates Improve Function of Endothelial Progenitor Cells from Human Peripheral Blood: Involvement of the Endogenous Antioxidant Defenses

Increased oxidative stress contributes to the functional impairment of endothelial progenitor cells (EPCs), the pivotal players in the servicing of the endothelial cell lining. Several evidences suggest that decreasing oxidative stress by natural compounds with antioxidant properties may improve EPCs bioactivity. Here, we investigated the effects of Lisosan G (LG), a Triticum Sativum grain powder, and Lady Joy (LJ), a bean lysate, on function of EPCs exposed to oxidative stress. Peripheral blood mononuclear cells were isolated and plated on fibronectin-coated culture dishes; adherent cells, identified as early EPCs, were pre-treated with different concentrations of LG and LJ and incubated with hydrogen peroxide (H₂O₂). Viability, senescence, adhesion, ROS production and antioxidant enzymes gene expression were evaluated. Lysate-mediated Nrf-2 (nuclear factor (erythroid-derived 2)-like 2)/ARE (antioxidant response element) activation, a modulator of oxidative stress, was assessed by immunocytochemistry. Lady Joy 0.35–0.7 mg/ml increases EPCs viability; pre-treatment with either LG 0.7 mg/ml and LJ 0.35–0.7 mg/ml protect EPCs viability against H₂O₂-induced injury. LG 0.7 and LJ 0.35–0.7 mg/ml improve EPCs adhesion; pre-treatment with either LG 0.35 and 0.7 mg/ml or LJ 0.35, 0.7 and 1.4 mg/ml preserve adhesiveness of EPCs exposed to H₂O₂. Senescence is attenuated in EPCs incubated with lysates 0.35 mg/ml. After exposure to H₂O₂, LG pre-treated cells show a lower senescence than untreated EPCs. Lysates significantly decrease H₂O₂-induced ROS generation. Both lysates increase glutathione peroxidase-1 and superoxide dismutase-2 (SOD-2) expression; upon H₂O₂ exposure, pre-treatment with LJ allows higher SOD-2 expression. Heme oxigenase-1 increases in EPCs pre-treated with LG even upon H₂O₂ exposure. Finally, incubation with LG 0.7 mg/ml results in Nrf-2 translocation into the nucleus both at baseline and after the oxidative challenge. Our data suggest a protective effect of lysates on EPCs exposed to oxidative stress through the involvement of antioxidant systems. Lisosan G seems to activate the Nrf-2/ARE pathways.     

Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling

The mammalian target of rapamycin complex 1(mTORC1) integrates diverse signals to control cell growth, proliferation, survival, and metabolism. Role of reactive oxygen species (ROS) on mTORC1 signaling remains obscure and mechanisms through which ROS modulate mTORC1 are not known. We demonstrate that low doses ROS exposure stimulate mTORC1 while high concentrations or long-term ROS treatment decrease mTORC1 activity in vivo and in a variety of cell lines. The dose/time needed for inhibition or activation are cell type-dependent. In HEK293 cells hydrogen peroxide (H₂O₂) stimulates phosphorylation of AMP-activated kinase (AMPK) (T172) and Raptor (S792), enhances association of activated AMPK with Raptor. Furthermore, AMPK inhibitor compound c inhibits H₂O₂-induced Raptor (S792) phosphorylation and reverses H₂O₂-induced de-phosphorylation of mTORC1 downstream targets p70-S6K1 (T389), S6 (S235/236) and 4E-BP1 (T37/46). H₂O₂ also stimulates association of endogenous protein phosphatase 2A catalytic subunit (PP2Ac) with p70-S6K1. Like compound c, inhibitor of PP2A, okadaic acid partially reverses inactivation of mTORC1 substrates induced by H₂O₂. Moreover, inhibition of PP2A and AMPK partially rescued cells from H₂O₂-induced cell death. High doses of H₂O₂ inhibit while low doses of H₂O₂ activate mTORC1 both in TSC2^?/? P53^?/? and TSC2^+/+ P53^?/? MEFs. These data suggest that PP2A and AMPK-mediated phosphorylation of Raptor mediate H₂O₂-induced inhibition of mTORC1 signaling.     

SK-N-MC Cell Death Occurs by Distinct Molecular Mechanisms in Response to Hydrogen Peroxide and Superoxide Anions: Involvements of JAK2-STAT3, JNK, and p38 MAP Kinases Pathways

Oxidative stress plays a vital role in the pathogenesis of neurodegenerative diseases. Nerve cells are incessantly exposed to environmental stresses leading to overproduction of some harmful species like reactive oxygen species (ROS). ROS including hydrogen peroxide and superoxide anion are potent inducers of various signaling pathways encompassing MAPKs and JAK-STAT pathways. In the current study, we scrutinized the effects of hydrogen peroxide and/or menadione (superoxide anion generator) on JNK/p38-MAPKs and JAK2-STAT3 pathways to elucidate the mechanism(s) by which each oxidant modulated the above-mentioned pathways leading to SK-N-MC cell death. Our results delineated that hydrogen peroxide and superoxide anion radical induced distinct responses as we showed that STAT3 and p38 were activated in response to hydrogen peroxide, but not superoxide anion radicals indicating the specificity in ROS-induced signaling pathways activations and behaviors. We also observed that menadione induced JNK-dependent p53 expression and apoptotic death in SK-N-MC cells while H₂O₂-induced JNK activation was p53 independent. Thus, we declare that ROS type has a key role in selective instigation of JNK/p38-MAPKs and JAK2-STAT3 pathways in SK-N-MC cells. Identifying these differential behaviors and mechanisms of hydrogen peroxide and superoxide anion functions illuminates the possible therapeutic targets in the prevention or treatment of ROS-induced neurodegenerative diseases such as Alzheimer’s disease.     

TAK1 activates AMPK-dependent cell death pathway in hydrogen peroxide-treated cardiomyocytes, inhibited by heat shock protein-70

The aim of this current study is to investigate the potential role of AMP-activated protein kinase (AMPK) in hydrogen peroxide (H₂O₂)-induced cardiomyocyte death, and focused on the signaling mechanisms of AMPK activation by H₂O₂. We observed a significant AMPK activation in H₂O₂-treated cardiomyocytes (both primary cells and H9c2 line). Inhibition of AMPK by its inhibitor or RNAi-reduced H₂O₂-induced cardiomyocyte death. We here proposed that transforming growth factor-β-activating kinase 1 (TAK1) might be the upstream kinase for AMPK activation by H₂O₂. H₂O₂-induced TAK1 activation, which recruited and activated AMPK. TAK1 inhibitor significantly suppressed H₂O₂-induced AMPK activation and following cardiomyocyte death, while over-expression of TAK1-facilitated AMPK activation and aggregated cardiomyocyte death. Importantly, heat shock protein-70 (HSP-70)-reduced H₂O₂-induced reactive oxygen species (ROS) accumulation, the TAK1/AMPK activation and cardiomyocyte death. In conclusion, we here suggest that TAK1 activates AMPK-dependent cell death pathway in H₂O₂-treated cardiomyocytes, and HSP-70 inhibits the signaling pathway by reducing ROS content.     

Torulene and torularhodin,protects human prostate stromal cells from hydrogen peroxide-induced oxidative stress damage through the regulation of Bcl-2/Bax mediated apoptosis

The current study was designed to elucidate the cytoprotective effects and possible mechanisms of torulene and torularhodin on hydrogen peroxide (H₂O₂)-induced oxidative stress damage in human prostate stromal cells (WPMY-1). After treated with H₂O₂, a notable decrease was appeared in cell viability, yet the decrease was attenuated when cells were pretreated with torulene and torularhodin (0.5–10?μM) as evaluated by WST-1 assay. Pretreatment with these two carotenoids significantly attenuated H₂O₂-induced apoptosis in WPMY-1 cells through the inhibition of intracellular reactive oxygen species (ROS) and malondialdehyde (MDA) overproduction, as well as the activation of the activities in catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px). Finally, pretreatment of cells with carotenoids resulted in the regulation of the mRNA and protein expression of Bcl-2 and Bax in H₂O₂-exposed prostate stromal cells. The present results indicate that both torulene and torularhodin can protect human prostate stromal cells from oxidative stress damage via Bcl-2/Bax mediated apoptosis.     

Hydrogen peroxide activation of ERK5 confers resistance to Jurkat cells against apoptosis induced by the extrinsic pathway

Reactive oxygen species (ROS) including hydrogen peroxide (H₂O₂) exhibit both pro-survival and pro-death signaling in leukemic cells. We examined the effect of exogenous H₂O₂ on Fas ligand (FasL) -induced apoptosis in Jurkat cells. H₂O₂ applied prior to (pre-conditioning) and during (post-conditioning) FasL stimulation attenuated early apoptosis through activation of EKR5. H₂O₂ increased the activated caspase-8 sequestered in the mitochondria thereby decreasing cell death through the extrinsic apoptotic pathway. In addition, inhibition of a protein tyrosine phosphatase likely explains the post-conditioning requirement for H₂O₂. Given that chemotherapeutic agents used for the treatment of acute lymphoblastic leukemia are thought to work partly through production of ROS, a simultaneous inhibition of the ERK5 pathway may abrogate the ROS-initiated pro-survival signaling for an enhanced cell kill.