Induction of adaptive response and enhancement of PC12 cell tolerance by 7-hydroxycholesterol and 15-deoxy-delta(12,14)-prostaglandin J2 through up-regulation of cellular glutathione via different mechanisms

Increasing evidence suggests an adaptive response induced by reactive oxygen species and other physiologically existing oxidative stimuli. We have recently reported that a variety of lipid peroxidation products at sublethal concentrations could induce adaptive response and enhance PC12 cell tolerance, although the detailed underlying molecular mechanisms have not been clearly clarified. In the present study, we found that both 7-hydroxycholesterol (7-OHCh) and 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) at sublethal concentrations significantly increased the cellular GSH as well as the enzyme activity of glutamate-cysteine ligase (GCL), the rate-limiting enzyme of GSH synthesis. Depletion of cellular GSH by buthionine sulfoximine completely abolished the adaptive response. Interestingly, treatment with 15d-PGJ2 significantly increased the gene expression of both subunits of GCL in an NF-E2-related factor 2 (Nrf2)-dependent manner, whereas neither 7-OHCh induced any considerable changes on the GCL gene expression nor did the Nrf2-small interfering RNA treatment exert any appreciable effects on the GSH elevation and subsequent adaptive response induced by 7-OHCh. These results demonstrate that the adaptive response induced by both 7-OHCh and 15d-PGJ2 is mediated similarly through the up-regulation of GSH but via different mechanisms.     

15-Deoxy-Delta12,14-prostaglandin J(2) protects against nitrosative PC12 cell death through up-regulation of intracellular glutathione synthesis

Nitrosative stress with subsequent inflammatory cell death has been associated with many neurodegenerative disorders. Expression of inducible nitric-oxide synthase and production of nitric oxide (NO) have been frequently elevated in many inflammatory disorders. NO can rapidly react with superoxide anion, producing more reactive peroxynitrite. In the present study, exposure of rat pheochromocytoma (PC12) cells to the peroxynitrite donor 3-morpholinosydnonimine hydrochloride (SIN-1) induced apoptosis, which accompanied depletion of intracellular glutathione (GSH), c-Jun N-terminal kinase activation, mitochondrial membrane depolarization, the cleavage of poly(ADP-ribose)polymerase, and DNA fragmentation. During SIN-1-induced apoptotic cell death, expression of inducible cyclooxygenase (COX-2), and peroxisome proliferator-activated receptor-gamma (PPARgamma) was elevated. SIN-1 treatment resulted in elevated production of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), an endogenous PPARgamma activator. Preincubation with 15d-PGJ(2) rendered PC12 cells resistant to nitrosative stress induced by SIN-1. 15d-PGJ(2) fortified an intracellular GSH pool through up-regulation of glutamylcysteine ligase, thereby preventing cells from SIN-1-induced GSH depletion. The above findings suggest that 15d-PGJ(2) may act as a survival mediator capable of augmenting cellular thiol antioxidant capacity through up-regulation of the intracellular GSH synthesis in response to the nitrosative insult.     

Accumulation of 15-deoxy-delta(12,14)-prostaglandin J2 adduct formation with Keap1 over time: effects on potency for intracellular antioxidant defence induction

The COX (cyclo-oxygenase) pathway generates the reactive lipid electrophile 15d-PGJ2 (15-deoxy-Delta(12,14)-prostaglandin J2), which forms covalent protein adducts that modulate cell signalling pathways. It has been shown that this regulates important biological responses, including protection against oxidative stress, and supports the proposal that 15d-PGJ2 has pharmacological potential. Protective pathways activated by 15d-PGJ2 include those controlling the synthesis of the intracellular antioxidants GSH and the enzyme HO-1 (haem oxygenase-1). The induction of the synthesis of these intracellular antioxidants is, in large part, regulated by covalent modification of Keap1 (Kelchlike erythroid cell-derived protein with 'capn'collar homologyassociated protein 1) by the lipid and the subsequent activation of the EpRE (electrophile-response element). For the first time, we show that the potency of 15d-PGJ2 as a signalling molecule in endothelial cells is significantly enhanced by the accumulation of the covalent adduct with 15d-PGJ2 and endogenous Keap1 over the time of exposure to the prostaglandin. The consequence of this finding is that signalling initiated by electrophilic lipids differs from agonists that do not form covalent adducts with proteins because the constant generation of very lowconcentrations of 15d-PGJ2 can lead to induction of GSH or HO-1. In the course of these studies we also found that a substantial amount (97-99%) of exogenously added 15d-PGJ2 is inactivated in the medium and does not enter the cells to initiate cell signalling. In summary, we propose that the accumulation of covalent adduct formation with signalling proteins provides a mechanism through which endogenous intracellular formation of electrophilic lipids from COX can exert an anti-inflammatory effect in vivo.     

Opposing roles for ERK1/2 in neuronal oxidative toxicity: distinct mechanisms of ERK1/2 action at early versus late phases of oxidative stress

Glutamate-induced oxidative toxicity is mediated by glutathione depletion in the HT22 mouse hippocampal cell line. Previous results with pharmacological agents implicated the extracellular signal-regulated kinases-1/2 (ERK1/2) in glutamate toxicity in HT22 cells and immature embryonic rat cortical neurons. In this report, we definitively establish a role for ERK1/2 in oxidative toxicity using dominant negative MEK1 expression in transiently transfected HT22 cells to block glutamate-induced cell death. In contrast, chronic activation of ERK (i.e. brought about by transfection of constitutively active ERK2 chimera) is not sufficient to trigger HT22 cell death demonstrating that ERK1/2 activation is not sufficient for toxicity. Activation of ERK1/2 in HT22 cells has a distinct kinetic profile with an initial peak occurring between 30 min and 1 h of glutamate treatment and a second peak typically emerging after 6 h. We demonstrate here that the initial phase of ERK1/2 induction is because of activation of metabotropic glutamate receptor type I (mGluRI). ERK1/2 activation by mGluRI contributes to an HT22 cell adaptive response to oxidative stress as glutamate-induced toxicity is enhanced upon pharmacological inhibition of mGluRI. The protective effect of ERK1/2 activation at early times after glutamate treatment is mediated by a restoration of glutathione (GSH) levels that are reduced because of depletion of intracellular cysteine pools. Thus, ERK1/2 appears to play dual roles in HT22 cells acting as part of a cellular adaptive response during the initial phases of glutamate-induced oxidative stress and contributing to toxicity during later stages of stress.     

15d-Deoxy-Delta12,14-prostaglandin J2 modulates collagen type I synthesis in human hepatic stellate cells by inducing oxidative stress

15 deoxy-Delta(12,14)-prostaglandin(2) (15d-PGJ(2)) is known to inhibit the proliferation of hepatic stellate cells (HSCs), major cellular components that cause hepatic fibrosis, in vitro. It also induces oxidative stress, which results in hepatic myofibroblast death. On the other hand, oxidative stress generally induces HSC proliferation and collagen synthesis in vitro, and liver fibrogenesis in vivo. In this study, we evaluated the effects of 15d-PGJ(2) at various concentrations on the viability and collagen synthesis of HSCs. 15d-PGJ(2) increased intracellular reactive oxygen species (ROS), and reduced the viability of human HSCs at concentrations 5 microM by inducing apoptotic cell death. In addition, the antioxidants alpha-tocopherol and N-acetylcysteine (NAC) blocked 15d-PGJ(2)-induced HSC death. Collagen I synthesis was increased 1.5-fold by 0.5 microM 15d-PGJ(2) treatment, but was reduced to 30% of the control level by 10 microM 15d-PGJ(2), and NAC pretreatment prevented these changes in collagen production by 15d-PGJ(2). We conclude that 15d-PGJ(2) may either induce or prevent hepatic fibrogenesis depending on its concentration.     

Thioredoxin as a molecular target of cyclopentenone prostaglandins

Prostaglandin (PG) D2, a major cyclooxygenase product in a variety of tissues and cells, readily undergoes dehydration to yield the bioactive cyclopentenone-type PGs of the J2 series, such as 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2). We have shown previously that 15d-PGJ2 is a potent electrophile that causes intracellular oxidative stress and redox alteration in human neuroblastoma SH-SY5Y cells. In the present study, based on the observation that the electrophilic center of 15d-PGJ2 was involved in the pro-oxidant effect, we investigated the role of thioredoxin 1 (Trx), an endogenous redox regulator, against 15d-PGJ2-induced oxidative cell injury. It was observed that the 15d-PGJ2-induced oxidative stress was significantly suppressed by the Trx overexpression. In addition, the treatment of SH-SY5Y cells with biotinylated 15d-PGJ2 resulted in the formation of a 15d-PGJ2-Trx adduct, indicating that 15d-PGJ2 directly modified the endogenous Trx in the cells. To further examine the mechanism of the 15d-PGJ2 modification of Trx, human recombinant Trx treated with 15d-PGJ2 was analyzed by mass spectrometry. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the 15d-PGJ2-treated human recombinant Trx demonstrated the addition of one molecule of 15d-PGJ2 per protein molecule. Moreover, the electrospray ionization-liquid chromatography/mass spectrometry/mass spectrometry analysis identified two cysteine residues, Cys-35 and Cys-69, as the targets of 15d-PGJ2. These residues may represent the direct sensors of the electrophilic PGs that induce the intracellular redox alteration and neuronal cell death.     

Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal,a key molecule in stress mediated signaling

Role of lipid peroxidation products, particularly 4-hydroxynonenal (4-HNE) in cell cycle signaling is becoming increasingly clear. In this article, recent studies suggesting an important role of 4-HNE in stress mediated signaling for apoptosis are critically evaluated. Evidence demonstrating the modulation of UV, oxidative stress, and chemical stress mediated apoptosis by blocking lipid peroxidation by the alpha-class glutathione S-transferases (GSTs) is presented which suggest an important role of these enzymes in protection against oxidative stress and a role of lipid peroxidation products in stress mediated signaling. Overexpression of 4-HNE metabolizing GSTs (mGSTA4-4, hGSTA4-4, or hGST5.8) protects cells against 4-HNE, oxidative stress (H(2)O(2) or xanthine/xanthine oxidase), and UV-A mediated apoptosis by blocking JNK and caspase activation suggesting a role of 4-HNE in the mechanisms of apoptosis caused by these stress factors. The intracellular concentration of 4-HNE appears to be crucial for the nature of cell cycle signaling and may be a determinant for the signaling for differentiation, proliferation, transformation, or apoptosis. The intracellular concentrations of 4-HNE are regulated through a coordinated action of GSTs (GSTA4-4 and hGST5.8) which conjugate 4-HNE to GSH to form the conjugate (GS-HNE) and the transporter 76 kDa Ral-binding GTPase activating protein (RLIP76), which catalyze ATP-dependent transport of GS-HNE. A mild stress caused by heat, UV-A, or H(2)O(2)with no apparent effect on the cells in culture causes a rapid, transient induction of hGST5.8 and RLIP76. These stress preconditioned cells acquire ability to metabolize and exclude 4-HNE at an accelerated pace and acquire relative resistance to apoptosis by UV and oxidative stress as compared to unconditioned control cells. This resistance of stress preconditioned cells can be abrogated by coating the cells with anti-RLIP76 antibodies which block the transport of GS-HNE. These studies and previous reports discussed in this article strongly suggest a key role of 4-HNE in stress mediated signaling.     

Adaptive response induced by lipid peroxidation products in cell cultures

The adaptive response induced by the lipid peroxidation products, such as phosphatidylcholine hydroperoxide, lysophosphatidylcholine (LysoPC), 15-deoxy-Delta(12,14)-prostaglandin J(2), 4-hydroxynonenal (4-HNE), hydroxyoctadecadienoic acid, 7-hydroxycholesterol, and cholesterol 5beta,6beta-epoxide, was investigated in this study. Although these products have been implicated in oxidative stress-related diseases, pretreatment with such compounds at sublethal concentrations significantly protected PC12 cells against subsequent oxidative stress induced by 6-hydroxydopamine. Moreover, 4-HNE and LysoPC also exhibited adaptive protection in human arterial endothelial cells. These findings suggest a general hormetic effect of such compounds in cell cultures and may lead to a reappraisal of the eventual role of reactive oxygen species and lipid peroxidation in organisms.     

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death ) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.     

Chebulinic acid attenuates glutamate-induced HT22 cell death by inhibiting oxidative stress,calcium influx and MAPKs phosphorylation

Glutamate-induced excitotoxicity and oxidative stress is a major causative factor in neuronal cell death in acute brain injuries and chronic neurodegenerative diseases. The prevention of oxidative stress is a potential therapeutic strategy. Therefore, in the present study, we aimed to examine a potential therapeutic agent and its protective mechanism against glutamate-mediated cell death. We first found that chebulinic acid isolated from extracts of the fruit of Terminalia chebula prevented glutamate-induced HT22 cell death. Chebulinic acid significantly reduced intracellular reactive oxygen species (ROS) production and Ca²⁺ influx induced by glutamate. We further demonstrated that chebulinic acid significantly decreased the phosphorylation of mitogen-activated protein kinases (MAPKs), including ERK1/2, JNK, and p38, as well as inhibiting pro-apoptotic Bax and increasing anti-apoptotic Bcl-2 protein expression. Moreover, we demonstrated that chebulinic acid significantly reduced the apoptosis induced by glutamate in HT22 cells. In conclusion, our results in this study suggest that chebulinic acid is a potent protectant against glutamate-induced neuronal cell death via inhibiting ROS production, Ca²⁺ influx, and phosphorylation of MAPKs, as well as reducing the ratio of Bax to Bcl-2, which contribute to oxidative stress-mediated neuronal cell death.     

Chloroquine inhibits glutamate-induced death of a neuronal cell line by reducing reactive oxygen species through sigma-1 receptor

Chloroquine, a widely used anti-malarial and anti-rheumatoid agent, has been reported to induce apoptotic and non-apoptotic cell death. Accumulating evidence now suggests that chloroquine can sensitize cancer cells to cell death and augment chemotherapy-induced apoptosis by inhibiting autophagy. However, chloroquine is reported to induce GM1 ganglioside accumulation in cultured cells at low μM concentrations and prevent damage to the blood brain barrier in mice. It remains unknown whether chloroquine has neuroprotective properties at concentrations below its reported ability to inhibit lysosomal enzymes and autophagy. In the present study, we demonstrated that chloroquine protected mouse hippocampal HT22 cells from glutamate-induced oxidative stress by attenuating production of excess reactive oxygen species. The concentration of chloroquine required to rescue HT22 cells from oxidative stress was much lower than that sufficient enough to induce cell death and inhibit autophagy. Chloroquine increased GM1 level in HT22 cells at low μM concentrations but glutamate-induced cell death occurred before GM1 accumulation, suggesting that GM1 induction is not related to the protective effect of chloroquine against glutamate-induced cell death. Interestingly, BD1047 and NE-100, sigma-1 receptor antagonists, abrogated the protective effect of chloroquine against glutamate-induced cell death and reactive oxygen species production. In addition, cutamesine (SA4503), a sigma-1 receptor agonist, prevented both glutamate-induced cell death and reactive oxygen species production. These findings indicate that chloroquine at concentrations below its ability to inhibit autophagy and induce cell death is able to rescue HT22 cells from glutamate-induced cell death by reducing excessive production of reactive oxygen species through sigma-1 receptors. These results suggest potential use of chloroquine, an established anti-malarial agent, as a neuroprotectant against oxidative stress, which occurs in a variety of neurodegenerative diseases.     

Arachidonic acid promotes glutamate-induced cell death associated with necrosis by 12- lipoxygenase activation in glioma cells

Glutamate induced glutathione (GSH) depletion in C6 rat glioma cells, which resulted in cell death. This cell death seemed to be apoptosis through accumulation of reactive oxygen species (ROS) or hydroperoxides representing cytochrome c release from mitochondria and internucleosomal DNA fragmentation. A significant increase of 12-lipoxygenase enzyme activity was observed in the presence of arachidonic acid (AA) under GSH depletion induced by glutamate. AA promoted the glutamate-induced cell death, which reduced caspase-3 activity and diminished internucleosomal DNA fragmentation. Furthermore, AA reduced intracellular NAD, ATP and membrane potentials, which indicated dysfunction of the mitochondrial membrane. Protease inhibitors such as N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3, 4-dichloroisocumarin (DCI) but no Ac-DEVD, a caspase inhibitor, suppressed the glutamate-induced cell death. AA reduced the inhibitory effect of TPCK and DCI on the glutamate-induced cell death. These results suggest that AA promotes cell death by inducing necrosis from caspase-3-independent apoptosis. This might occur through lipid peroxidation initiated by ROS or lipid hydroperoxides generated during GSH depletion in C6 cells.     

Role of 4-hydroxynonenal in stress-mediated apoptosis signaling

In this mini review we summarize recent studies from our laboratory, which show the involvement of 4-hydroxynonenal (4-HNE) in cell cycle signaling. We demonstrate 4-HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase and caspase-3 activation. Cells exposed to mild, transient, heat or oxidative stress acquire capacity to exclude intracellular 4-HNE at a faster rate by inducing hGST5.8 which conjugate 4-HNE to GSH, and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4-HNE. The cells preconditioned with mild transient stress acquire resistance to H(2)O(2) and 4-HNE induced apoptosis by excluding intracellular 4-HNE at an accelerated pace. Furthermore, a decrease in intracellular concentration of 4-HNE achieved by transfecting cells with mGSTA4-4 or hGSTA4-4 results in a faster growth rate. These studies strongly suggest a role of 4-HNE in stress mediated signaling.     

Characterization of 4-hydroxy-2-nonenal metabolism in stellate cell lines derived from normal and cirrhotic rat liver

During oxidative stress, reactive aldehydes, including trans-4-hydroxy-2-nonenal (4-HNE), are generated by peroxidation of membrane lipids and purportedly stimulate hepatic stellate cells to produce excessive extracellular matrix, including type I collagen. An important question concerning the ability of 4-HNE to modulate collagen production by stellate cells is the potential of these specialized cells to detoxify 4-HNE. The objective of the present study was to characterize the ability of stellate cell lines, derived from normal (NFSC) and cirrhotic (CFSC) rat livers, to metabolize 4-HNE by oxidative, reductive and conjugative pathways. These two stellate cell lines were noted to have differing susceptibilities to the cytotoxic effect of 4-HNE. Treatment of both stellate cell lines with a range of 4-HNE doses demonstrated that the concentration which was cytotoxic to 50% of CFSC (TD(50)) was 25% greater than that for NFSC (967.57+/-9.26 nmol/10(6) cells vs. 769.90+/-5.32 nmol/10(6) cells respectively). The capacity of these cell lines to metabolizes 4-HNE was determined by incubating them in suspension with 50 microM 4-HNE (10 nmol/10(6) cell); 4-HNE elimination and metabolite formation were quantified over a 20 min time course. Both stellate cell lines rapidly metabolized 4-HNE, with the CFSC line eliminating 4-HNE at a rate that was approx. 2-fold greater than the NFSC line. The rate of 4-HNE metabolism attributable to glutathione S-transferase (GST) was similar in both cell lines, though differential cell specific expressions of GST isoforms GSTP1-1 and GSTA4-4 were observed. The greater rate of 4-HNE elimination by CFSC was attributable to its aldehyde dehydrogenase (ALDH) activity which accounted for approx. 50% of 4-HNE metabolism in CFSC but was insignificant in NFSC. Neither cell line had detectable alcohol dehydrogenase activity or protein levels. Measurement of cellular GSH concentrations revealed that NFSC contain approx. 2-fold greater concentrations of GSH when compared to CFSC and that following 4-HNE treatment, GSH levels were rapidly depleted from both cell lines. Concomitant with 4-HNE mediated GSH depletion, a corresponding increase in the 4-HNE-glutathione adduct formation was observed with the NFSC line forming greater amounts of the glutathione adduct than did the CFSC line. Taken together, these data demonstrate that both stellate cell lines have the capacity to metabolize 4-HNE but that CFSC have a greater rate of metabolism which is attributable to their greater ALDH activity, suggesting that the stellate cells isolated from cirrhotic liver may be differentially responsive to the biologic effects of 4-HNE.     

Resveratrol protects against 4-HNE induced oxidative stress and apoptosis in Swiss 3T3 fibroblasts

Here we report on the marked protective effect of resveratrol on 4-hydroxynonenal (4-HNE) induced oxidative stress and apoptotic death in Swiss 3T3 fibroblasts. 4-HNE, one of the major aldehydic products of the peroxidation of membrane w-6 polyunsaturated fatty acids, has been suggested to contribute to oxidant stress mediated cell injury. Indeed, in vitro treatment of 3T3 fibroblasts with 4-HNE induced a condition of oxidative stress as monitored by the oxidation of dichlorofluorescein diacetate; this reaction was prevented when cells were pretreated with resveratrol. Further, 4-HNE-treated fibroblasts eventually underwent apoptotic death as determined by differential staining and internucleosomal DNA fragmentation. Resveratrol pretreatment also prevented 4-HNE induced DNA fragmentation and apoptosis. These observations are consistent with a potential role of lipid peroxidation-derived products in programmed cell death and demonstrate that resveratrol can counteract this effect by quenching cell oxidative stress.