Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.     

Reactive nitrogen species-induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase

Nitrogen dioxide is a highly toxic reactive nitrogen species (RNS) recently discovered as an inflammatory oxidant with great potential to damage tissues. We demonstrate here that cell death by RNS was caused by c-Jun N-terminal kinase (JNK). Activation of JNK by RNS was density dependent and caused mitochondrial depolarization and nuclear condensation. JNK activation by RNS was abolished in cells lacking functional Fas or following expression of a truncated version of Fas lacking the intracellular death domain. In contrast, RNS induced JNK potently in cells expressing a truncated version of tumor necrosis factor receptor 1 or cells lacking tumor necrosis factor receptor 1 (TNF-R1), illustrating a dependence of Fas but not TNF-R1 in RNS-induced signaling to JNK. Furthermore, Fas was oxidized, redistributed, and colocalized with Fas-associated death domain (FADD) in RNS-exposed cells, illustrating that RNS directly targeted Fas. JNK activation and cell death by RNS occurred in a Fas ligand- and caspase-independent manner. While the activation of JNK by RNS or FasL required FADD, the cysteine-rich domain 1 containing preligand assembly domain required for FasL signaling was not involved in JNK activation by RNS. These findings illustrate that RNS cause cell death in a Fas- and JNK-dependent manner and that this occurs through a pathway distinct from FasL. Thus, avenues aimed at preventing the interaction of RNS with Fas may attenuate tissue damage characteristic of chronic inflammatory diseases that are accompanied by high levels of RNS.     

Interplay of Cu,Zn superoxide dismutase and nitric oxide synthase in neurodegenerative processes

Reactive oxygen and nitrogen species (ROS and RNS) have been extensively recognized as important signaling molecules implicated in physiological processes such as gene expression, cell differentiation and immune activation. Nevertheless, continuous production of these species may produce oxidative and/or nitrosative stress resulting in cell damage and ultimately leading to cell death. Due to the high oxygen consumption and relative poor antioxidant defense, the central nervous system is highly susceptible to ROS- and RNS-mediated toxicity. Actually, the oxidative and nitrosative stress have been implicated in the pathogenesis of neurodegeneration of a large variety of neurological disorders. This review will cover some aspects of the involvement of ROS- and RNS-mediated apoptotic processes occurring in cellular models of familial amyotrophic lateral sclerosis (FALS), in particular the cases associated with mutations in SOD1, the gene encoding Cu,Zn superoxide dismutase (Cu,Zn SOD). A possible role for proteasome in the inhibition of neurodegenerative process by balancing ROS and RNS species is envisaged on the basis of evidence provided by results obtained from studies on this experimental model.     

Reactive oxygen species mediate crosstalk between NF-kappaB and JNK

The activation of NF-kappaB inhibits apoptosis via a mechanism involving upregulation of various antiapoptotic genes, such as cellular FLICE-inhibitory protein (c-FLIP), Bcl-xL, A1/Bfl-1, and X chromosome-liked inhibitor of apoptosis (XIAP). In contrast, the activation of c-Jun N-terminal kinase (JNK) promotes apoptosis in a manner that is dependent on the cell type and the context of the stimulus. Recent studies have indicated that one of the antiapoptotic functions of NF-kappaB is to downregulate JNK activation. Further studies have also revealed that NF-kappaB inhibits JNK activation by suppressing accumulation of reactive oxygen species (ROS). In this review, we will focus on the signaling crosstalk between the NF-kappaB and JNK cascades via ROS.     

Oxidative depolymerization of polysaccharides by reactive oxygen/nitrogen species

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are constantly produced and are tightly regulated to maintain a redox balance (or homeostasis) together with antioxidants (e.g. superoxide dismutase and glutathione) under normal physiological circumstances. These ROS/RNS have been shown to be critical for various biological events including signal transduction, aging, apoptosis, and development. Despite the known beneficial effects, an overproduction of ROS/RNS in the cases of receptor-mediated stimulation and disease-induced oxidative stress can inflict severe tissue damage. In particular, these ROS/RNS are capable of degrading macromolecules including proteins, lipids and nucleic acids as well as polysaccharides, and presumably lead to their dysfunction. The purpose of this review is to highlight (1) chemical mechanisms related to cell-free and cell-based depolymerization of polysaccharides initiated by individual oxidative species; (2) the effect of ROS/RNS-mediated depolymerization on the successive cleavage of the glycosidic linkage of polysaccharides by glycoside hydrolases; and (3) the potential biological outcome of ROS/RNS-mediated depolymerization of polysaccharides.     

Oxidative and nitrosative signaling in plants: Two branches in the same tree?

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) constitute key features underpinning the dynamic nature of cell signaling systems in plants. Despite their importance in many aspects of cell biology, our understanding of oxidative and especially of nitrosative signaling and their regulation remains poorly understood. Early reports have established that ROS and RNS coordinately regulate plant defense responses to biotic stress. In addition, evidence has accumulated demonstrating that there is a strong cross-talk between oxidative and nitrosative signaling upon abiotic stress conditions. The goal of this mini-review is to provide latest findings showing how both ROS and RNS comprise a coordinated oxidative and nitrosative signaling network that modulates cellular responses in response to environmental stimuli.Key words: abiotic stress, nitrosative stress, oxidative stress, reactive nitrogen species, reactive oxygen species, signaling     

Redox control of cell fate by MAP kinase: physiological roles of ASK1-MAP kinase pathway in stress signaling

The intracellular redox state is a key determinant of cell fate, such as cell survival, proliferation, differentiation, and apoptosis. Redox imbalance is closely linked to a variety of human diseases, so that the intracellular redox condition should be tightly regulated. The redox state of the cell is a consequence of the precise balance between the levels of oxidizing and reducing equivalents, such as reactive oxygen species (ROS) and endogenous antioxidants. ROS are not only toxicants to the cell, but also second messengers in intracellular signal transduction, and control the action of several signaling pathways, including mitogen-activated protein (MAP) kinases. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase of the c-Jun N-terminal kinase (JNK) and p38 MAP kinase pathways, which is preferentially activated in response to various types of stress such as oxidative stress and plays pivotal roles in a wide variety of cellular responses. Recent studies have revealed that ASK1 is also required for innate immune response through ROS production. In this review, we focus on redox control of cell function by MAP kinase signaling, and provide the advanced mechanism of redox-regulated ASK1 activation and physiological roles of the ASK1-MAP kinase pathway in stress signaling.     

Redox proteomics: basic principles and future perspectives for the detection of protein oxidation in plants

The production and scavenging of chemically reactive species, such as ROS/RNS, are central to a broad range of biotic and abiotic stress and physiological responses in plants. Among the techniques developed for the identification of oxidative stress-induced modifications on proteins, the so-called 'redox proteome', proteomics appears to be the best-suited approach. Oxidative or nitrosative stress leaves different footprints in the cell in the form of different oxidatively modified components and, using the redox proteome, it will be possible to decipher the potential roles played by ROS/RNS-induced modifications in stressed cells. The purpose of this review is to present an overview of the latest research endeavours in the field of plant redox proteomics to identify the role of post-translational modifications of proteins in developmental cell stress. All the strategies set up to analyse the different oxidized/nitrosated amino acids, as well as the different reactivities of ROS and RNS for different amino acids are revised and discussed. A growing body of evidence indicates that ROS/RNS-induced protein modifications may be of physiological significance, and that in some cellular stresses they may act causatively and not arise as a secondary consequence of cell damage. Thus, although previously the oxidative modification of proteins was thought to represent a detrimental process in which the modified proteins were irreversibly inactivated, it is now clear that, in plants, oxidatively/nitrosatively modified proteins can be specific and reversible, playing a key role in normal cell physiology. In this sense, redox proteomics will have a central role in the definition of redox molecular mechanisms associated with cellular stresses.     

Mitochondrial c-Jun N-terminal kinase (JNK) signaling initiates physiological changes resulting in amplification of reactive oxygen species generation

The JNK signaling cascade is critical for cellular responses to a variety of environmental and cellular stimuli. Although gene expression aspects of JNK signal transduction are well studied, there are minimal data on the physiological impact of JNK signaling. To bridge this gap, we investigated how JNK impacted physiology in HeLa cells. We observed that inhibition of JNK activity and JNK silencing with siRNA reduced the level of reactive oxygen species (ROS) generated during anisomycin-induced stress in HeLa cells. Silencing p38 had no significant impact on ROS generation under anisomycin stress. Moreover, JNK signaling mediated amplification of ROS production during stress. Mitochondrial superoxide production was shown to be the source of JNK-induced ROS amplification, as an NADPH oxidase inhibitor demonstrated little impact on JNK-mediated ROS generation. Using mitochondrial isolation from JNK null fibroblasts and targeting the mitochondrial scaffold of JNK, Sab, we demonstrated that mitochondrial JNK signaling was responsible for mitochondrial superoxide amplification. These results suggest that cellular stress altered mitochondria, causing JNK to translocate to the mitochondria and amplify up to 80% of the ROS generated largely by Complex I. This work demonstrates that a sequence of events exist for JNK mitochondrial signaling whereby ROS activates JNK, thereby affecting mitochondrial physiology, which can have effects on cell survival and death.     

Superoxide activates mTOR–eIF4E–Bax route to induce enhanced apoptosis in leukemic cells

Mammalian target of rapamycin (mTOR) is a central kinase that regulates cell survival, proliferation and translation. Reactive oxygen species (ROS) are second messengers with potential in manipulating cellular signaling. Here we report that two ROS generating phytochemicals, hydroxychavicol and curcumin synergize in leukemic cells in inducing enhanced apoptosis by independently activating both mitogen activated protein kinase (MAPK) (JNK and P³⁸) and mTOR pathways. Low level transient ROS generated after co-treatment with these phytochemicals led to activation of these two pathways. Both mTOR and MAPK pathways played important roles in co-treatment-induced apoptosis, by knocking down either mTOR or MAPKs inhibited apoptosis. Activation of mTOR, as evident from phosphorylation of its downstream effector eukaryotic translation initiation factor 4E-binding protein 1, led to release of eukaryotic translation initiation factor 4E (eIF4E) which was subsequently phosphorylated by JNK leading to translation of pro-apoptotic proteins Bax and Bad without affecting the expression of anti-apoptotic protein Bcl-xl. Our data suggest that mTOR and MAPK pathways converge at eIF4E in co-treatment-induced enhanced apoptosis and provide mechanistic insight for the role of mTOR activation in apoptosis.     

An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway

Inhibition of NF-kappaB activation increases susceptibility to tumor necrosis factor (TNF)alpha-induced cell death, concurrent with caspases and prolonged c-Jun N-terminal kinase (JNK) activation, and reactive oxygen species (ROS) accumulation. However, the detailed mechanisms are unclear. Here we show that cellular FLICE-inhibitory protein (c-FLIP) is rapidly lost in NF-kappaB activation-deficient, but not wild-type fibroblasts upon TNFalpha stimulation, indicating that NF-kappaB normally maintains the cellular levels of c-FLIP. The ectopic expression of the long form of c-FLIP (c-FLIPL) inhibits TNFalpha-induced prolonged JNK activation and ROS accumulation in NF-kappaB activation-deficient fibroblasts. Conversely, TNFalpha induces prolonged JNK activation and ROS accumulation in c-Flip-/- fibroblasts. Moreover, c-FLIPL directly interacts with a JNK activator, MAP kinase kinase (MKK)7, in a TNFalpha-dependent manner and inhibits the interactions of MKK7 with MAP/ERK kinase kinase 1, apoptosis-signal-regulating kinase 1, and TGFbeta-activated kinase 1. This stimuli-dependent interaction of c-FLIPL with MKK7 might selectively suppress the prolonged phase of JNK activation. Taken that ROS promote JNK activation and activation of the JNK pathway may promote ROS accumulation, c-FLIPL might block this positive feedback loop, thereby suppressing ROS accumulation.     

Regulation of necrosis of H9c2 myogenic cells upon transient energy deprivation. Rapid deenergization of mitochondria precedes necrosis and is controlled by reactive oxygen species, stress kinase JNK, HSP72 and ARC

Subjecting myogenic H9c2 cells to transient energy deprivation leads to a caspase-independent death with typical features of necrosis. Here we show that the rupture of cytoplasmic membrane, the terminal event in necrosis, is shortly preceded by rapid depolarization of mitochondrial membranes. The rapid deenergization of mitochondria critically depended upon prior generation of reactive oxygen species (ROS) during ATP depletion stage. Accordingly, expression of catalase prevented mitochondrial depolarization and averted subsequent necrosis. Interestingly, trifluoperazine, a compound that protects cells from ischemic insults, prevented necrosis of H9c2 cells through inhibition of ROS production. Other factors that regulated the mitochondrial membrane depolarization and subsequent loss of plasma membrane integrity include a stress kinase JNK activated at early steps of recovery from ATP depletion, as well as an apoptotic inhibitory protein ARC. Accordingly, inhibition of JNK or overexpression of ARC prevented mitochondrial depolarization and rescued H9c2 cells from necrosis. ROS and JNK affected mitochondrial deenergization and necrosis independently of each other since inhibition of ROS production did not prevent activation of JNK, whereas inhibition of JNK did not suppress ROS accumulation. Therefore, JNK activation and ROS production represent two independent pathways that control mitochondrial depolarization and subsequent necrosis of cells subjected to transient energy deprivation. Overexpression of ARC, although preventing mitochondrial depolarization, did not affect either JNK activation or production of ROS. The major heat shock protein Hsp72 inhibited JNK-related steps of necrotic pathway but did not affect ROS accumulation. Interestingly, mitochondrial depolarization and subsequent necrosis can be suppressed by an Hsp72 mutant Hsp72DeltaEEVD, which lacks chaperone function but can efficiently suppress JNK activation. Thus, Hsp72 is directly implicated in a signaling pathway, which leads to necrotic death.     

Cellular events involved in butyric acid-induced T cell apoptosis

We have previously demonstrated that butyric acid induces cytotoxicity and apoptosis of murine thymocytes, splenic T cells, and human Jurkat T cells. Therefore, to determine the apoptotic signaling pathway induced by butyric acid, we investigated the contribution of reactive oxygen species (ROS), mitochondria, ceramide, and mitogen-activated protein kinases in butyric acid-induced human Jurkat cell apoptosis. After exposure of cells to butyric acid, a pronounced accumulation of ROS was seen. Pretreatment of cells with the antioxidant N-acetyl-cysteine or 3-aminobenzamide attenuated butyric acid-induced apoptosis through a reduction of ROS generation. Cytochrome c, apoptosis-inducing factor, and second mitochondria-derived activator of caspases protein release from mitochondria into the cytosol were detected shortly after butyric acid treatment. Exposure of cells to butyric acid resulted in an increase in cellular ceramide in a time-dependent fashion. In addition, butyric acid-induced apoptosis was inhibited by DL-threo-dihidrosphingosine, a potent inhibitor of sphingosine kinase. Using anti-extracellular signal-regulated kinase (ERK), anti-c-Jun N-terminal kinase (JNK), and anti-p38 phosphospecific Abs, we showed a decrease in ERK, but not in JNK and p38 phosphorylation after treatment of cells with butyric acid. Pretreatment of cells with the JNK inhibitor SP600125 attenuated the effect of butyric acid on apoptosis, whereas no effect was seen with the p38 inhibitor SB202190 or the ERK inhibitor PD98059. Taken together, our results indicate that butyric acid-induced T cell apoptosis is mediated by ceramide production, ROS synthesis in mitochondria, and JNK activation in the mitogen-activated protein kinase cascade. Finally, these results were further substantiated by the expression profile of butyric acid-treated Jurkat cells obtained by means of cDNA array.     

Piceatannol attenuates hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells by blocking down-regulation of Bcl-XL and activation of JNK

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.     

ASK Family Proteins in Stress Response and Disease

Cells are continuously exposed to reactive oxygen species (ROS) generated by aerobic metabolism. Excessively generated ROS causes severe dysfunctions to cells as oxidative stress. On the other hand, there is increasing evidence that ROS plays important roles as a signaling intermediate that induces a wide variety of cellular responses such as proliferation, differentiation, senescence, and apoptosis. To transmit physiological ROS-mediated signals and to adapt to oxidative stress, cells are equipped with various intracellular signal transduction systems, represented by mitogen-activated protein kinase (MAPK) cascades. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream regulator of the stress-activated MAPK cascades and has been shown to play critical roles in ROS-mediated cellular responses. Here, we highlight the roles of members of the ASK family, which consists of ASK1 and newly characterized ASK2, in ROS signaling with their possible involvement in human diseases.     

The benefits of oxidative stress for tissue repair and regeneration

Recent work has strengthened Drosophila imaginal discs as a model system for regeneration studies. Evidence is accumulating that oxidative stress drives the cellular responses for repair and regeneration. Drosophila imaginal discs generate a burst of reactive oxygen species (ROS) upon damage that is necessary for the activation of the Jun N-terminal kinase (JNK) and p38 MAP kinase signaling pathways. Moreover, these pathways are pivotal in the activation of regenerative growth. A hypothetical mechanism of how the ROS are initiated, and how repair and regeneration is activated is discussed here.     

Functional complementation in yeast reveals a protective role of chloroplast 2-Cys peroxiredoxin against reactive nitrogen species

The importance of nitric oxide (NO) as a signaling molecule to various plant physiological and pathophysiological processes is becoming increasingly evident. However, little is known about how plants protect themselves from nitrosative and oxidative damage mediated by NO and NO-derived reactive nitrogen species (RNS). Peroxynitrite, the product of the reaction between NO and superoxide anion, is considered to play a central role in RNS-induced cytotoxicity, as a result of its potent ability to oxidize diverse biomolecules. Employing heterologous expression in bacteria and yeast, we investigated peroxynitrite-scavenging activity in plants of 2-Cys peroxiredoxin (2CPRX), originally identified as a hydroperoxide-reducing peroxidase that is ubiquitously distributed among organisms. The putative mature form of a chloroplast-localized 2CPRX from Arabidopsis thaliana was overproduced in Escherichia coli as an amino-terminally hexahistidine-tagged fusion protein. The purified recombinant 2CPRX, which was catalytically active as peroxidase, efficiently prevented the peroxynitrite-induced oxidation of a sensitive compound. We also examined in vivo the ability of the Arabidopsis 2CPRX to complement the 2CPRX deficiency of a Saccharomyces cerevisiae mutant. Functional expression in the mutant strain of the Arabidopsis 2CPRX not only increased cellular tolerance to hydrogen peroxide, but also complemented the hypersensitive growth defect induced by nitrite-mediated cytotoxicity. The complemented cells significantly enhanced the capacity to reduce RNS-mediated oxidative damages. The results presented here demonstrate a new role of plant 2CPRX as a critical determinant of the resistance to RNS, and support the existence of a plant enzymatic basis for RNS metabolism.