The role of human glutathione S-transferases hGSTA1-1 and hGSTA2-2 in protection against oxidative stress.

In order to elucidate the protective role of glutathione S-transferases (GSTs) against oxidative stress, we have investigated the kinetic properties of the human alpha-class GSTs, hGSTA1-1 and hGSTA2-2, toward physiologically relevant hydroperoxides and have studied the role of these enzymes in glutathione (GSH)-dependent reduction of these hydroperoxides in human liver. We have cloned hGSTA1-1 and hGSTA2-2 from a human lung cDNA library and expressed both in Escherichia coli. Both isozymes had remarkably high peroxidase activity toward fatty acid hydroperoxides, phospholipid hydroperoxides, and cumene hydroperoxide. In general, the activity of hGSTA2-2 was higher than that of hGSTA1-1 toward these substrates. For example, the catalytic efficiency (kcat/Km) of hGSTA1-1 for phosphatidylcholine (PC) hydroperoxide and phosphatidylethanolamine (PE) hydroperoxide was found to be 181.3 and 199.6 s-1 mM-1, respectively, while the catalytic efficiency of hGSTA2-2 for PC-hydroperoxide and PE-hydroperoxide was 317.5 and 353 s-1 mM-1, respectively. Immunotitration studies with human liver extracts showed that the antibodies against human alpha-class GSTs immunoprecipitated about 55 and 75% of glutathione peroxidase (GPx) activity of human liver toward PC-hydroperoxide and cumene hydroperoxide, respectively. GPx activity was not immunoprecipitated by the same antibodies from human erythrocyte hemolysates. These results show that the alpha-class GSTs contribute a major portion of GPx activity toward lipid hydroperoxides in human liver. Our results also suggest that GSTs may be involved in the reduction of 5-hydroperoxyeicosatetraenoic acid, an important intermediate in the 5-lipoxygenase pathway.     

Glutathione S-transferases as antioxidant enzymes: small cell lung cancer (H69) cells transfected with hGSTA1 resist doxorubicin-induced apoptosis

It has been suggested that the alpha-class glutathione S-transferases (GSTs) protect various cell types from oxidative stress and lipid peroxidation (LPO). In order to examine the protective role of alpha-class GST isozyme hGSTA1-1 against doxorubicin (DOX)-induced lipid peroxidation, cytotoxicity, and apoptosis, human small cell lung cancer (SCLC) H69 cells were stably transfected with hGSTA1. Immunological and biochemical characterization of hGSTA1-transfected cells revealed the expression of functionally active hGSTA1-1 localized near the cellular plasma membranes. hGSTA1-transfected cells acquired significantly increased resistance to the DOX-induced cytotoxicity by suppressing lipid peroxidation levels in these cells. Overexpression of hGSTA1-1 in cells inhibited DOX-mediated depletion of GSH and higher GSH levels were found in DOX-treated hGSTA1-transfected cells as compared with empty vector-transfected controls. hGSTA1-1 overexpression also provided protection to cells from DOX-induced apoptosis by inhibiting phosphorylation of c-Jun-N-terminal kinases (JNK), caspase-3 activation, and by preserving the levels of anti-apoptotic protein Bcl-2. These results are consistent with the idea that the alpha-class GSTs provide protection against oxidative stress by attenuating lipid peroxidation and these enzymes can modulate signaling for apoptosis.     

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.     

Regulation of 4-hydroxynonenal-mediated signaling by glutathione S-transferases

4-Hydroxynonenal (HNE), one of the major end products of lipid peroxidation, has been shown to be involved in signal transduction and available evidence suggests that it can affect cell cycle events in a concentration-dependent manner. Glutathione S-transferases (GSTs) can modulate the intracellular concentrations of HNE by affecting its generation during lipid peroxidation by reducing hydroperoxides and also by converting it into a glutathione conjugate. We have recently demonstrated that overexpression of the Alpha class GSTs in cells leads to lower steady-state levels of HNE, and these cells acquire resistance to apoptosis induced by lipid peroxidation-causing agents such as H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics, suggesting that signaling for apoptosis by these agents is transduced through HNE. Cells with the capacity to exclude HNE from the intracellular environment at a faster rate are relatively more resistant to apoptosis caused by H(2)O(2), UVA, superoxide anion, and pro-oxidant xenobiotics as well as by HNE, suggesting that HNE may be a common denominator in mechanisms of apoptosis caused by oxidative stress. We have also shown that transfection of adherent cells with HNE-metabolizing GSTs leads to transformation of these cells due to depletion of HNE. These recent studies from our laboratories, which strongly suggest that HNE is a key signaling molecule and that GSTs, being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.     

Role of glutathione S-transferases in oxidative stress-induced male germ cell apoptosis

Cellular apoptosis in a tissue may occur for the maintenance of proper ratio of cells or because of toxic effects of free radicals or other agents. Male germ cell apoptosis is pivotal in maintaining the proper functioning of the testis, but it is not clear how free radicals affect germ cells and what the defense mechanisms are that are used by these cells to combat the toxic effects of the products of oxidative stress. This study shows that male germ cells are susceptible to H(2)O(2)-induced stress and, upon exposure to H(2)O(2) in vitro, demonstrate a typical apoptotic phenotype that includes DNA fragmentation and formation of DNA ladders. Other changes include considerable accumulation of products of lipid peroxidation in the germ cells after exposure to H(2)O(2). Evidence is presented for the existence of multiple isoforms of glutathione S-transferases (GSTs) that possess both transferase and Se-independent peroxidase activity. Germ cell GST activity increases after H(2)O(2) exposure. If this increase in activity is inhibited with suitable inhibitors, the formation of products of lipid peroxidation is augmented, resulting in germ cell apoptosis. Also, when constitutive GST activity is inhibited, accumulation of products of lipid peroxidation occurs, resulting in increased cellular apoptosis. These data show that GSTs form a part of adaptive response of germ cells to oxidative stress and are important constituents in detoxifying the products of lipid peroxidation.     

Human GSTA1-1 reduces c-Jun N-terminal kinase signalling and apoptosis in Caco-2 cells

The effect of GSTA1-1 (glutathione S-transferase Alpha 1-1) on JNK (c-Jun N-terminal kinase) activation was investigated in Caco-2 cells in which GSTA1 expression increases with degree of confluency, and in MEF3T3 cells with Tet-Off-inducible GSTA1 expression. Comparison of GSTA1 expression in pre-confluent, confluent and 8-day post-confluent Caco-2 cells revealed progressively increasing mRNA and protein levels at later stages of confluency. Exposure of pre-confluent cells to stress conditions including IL-1beta (interleukin-1beta), H2O2 or UV irradiation resulted in marked increases in JNK activity as indicated by c-Jun phosphorylation. However, JNK activation was significantly reduced in post-confluent cells exposed to the same stresses. Western-blot analysis of GSTA1-1 protein bound to JNK protein pulled down from cellular extracts showed approx. 4-fold higher GSTA1-1-JNK complex formation in post-confluent cells compared with pre-confluent cells. However, stress conditions did not alter the amount of GSTA1-1 bound to JNK. The role of GSTA1-1 in JNK suppression was more specifically revealed in Tet-Off-inducible MEF3T3-GSTA1-1 cells in which GSTA1 overexpression significantly reduced phosphorylation of c-Jun following exposure to IL-1beta, H2O2 and UV irradiation. Finally, the incidence of tumour necrosis factor alpha/butyrate-induced apoptosis was significantly higher in pre-confluent Caco-2 cells expressing low levels of GSTA1 compared with post-confluent cells. These results indicate that GSTA1 suppresses activation of JNK signalling by a pro-inflammatory cytokine and oxidative stress and suggests a protective role for GSTA1-1 in JNK-associated apoptosis.     

Transfection with 4-hydroxynonenal-metabolizing glutathione S-transferase isozymes leads to phenotypic transformation and immortalization of adherent cells.

4-Hydroxy-2-trans-nonenal (4-HNE), one of the major end products of lipid peroxidation, has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. We show for the first time that incorporation of 4-HNE-metabolizing glutathione S-transferase (GST) isozyme, hGSTA4-4, into adherent cell lines HLE B-3 and CCL-75, by either cDNA transfection or microinjection of active enzyme, leads to their transformation. The dramatic phenotypic changes due to the incorporation of hGSTA4-4 include rounding of cells and anchorage-independent rapid proliferation of immortalized, rounded, and smaller cells. Incorporation of the inactive mutant of hGSTA4-4 (Y212F) in cells by either microinjection or transfection does not cause transformation, suggesting that the activity of hGSTA4-4 toward 4-HNE is required for transformation. This is further confirmed by the fact that mouse and Drosophila GST isozymes (mGSTA4-4 and DmGSTD1-1), which have high activity toward 4-HNE and subsequent depletion of 4-HNE, cause transformation whereas human GST isozymes hGSTP1-1 and hGSTA1-1, with minimal activity toward 4-HNE, do not cause transformation. In cells overexpressing active hGSTA4-4, expression of transforming growth factor beta1, cyclin-dependent kinase 2, protein kinase C betaII and extracellular signal regulated kinase is upregulated, whereas expression of p53 is downregulated. These studies suggest that alterations in 4-HNE homeostasis can profoundly affect cell-cycle signaling events.     

Modulation of GST P1-1 activity by polymerization during apoptosis

Glutathione S-transferases (GSTs, EC 2.5.1.18) belong to a large family of functionally different enzymes that catalyze the S-conjugation of glutathione with a wide variety of electrophilic compounds including carcinogens and anticancer drugs. Drug resistance may result from reduction in apoptosis of neoplastic cells when exposed to antineoplastic drugs. The c-Jun N-terminal Kinase (JNK) belongs to the family of stress kinases and has been shown to be required for the maximal induction of apoptosis by DNA-damaging agents. Recently, an inhibition of JNK activity by GST P1-1, which was reversed by polymerization induced by oxidative stress, has been reported in 3T3-4A mouse fibroblast cell lines. The finding that GST P1-1 might inhibit JNK activity and that it is frequently highly expressed in tumor tissues suggests its possible implication in "apoptosis resistance" during antineoplastic therapy. We investigated the modulation of GST P1-1 during apoptosis in a neoplastic T-cell line (Jurkat) induced by hydrogen peroxide and etoposide. Apoptosis was paralleled by the appearance of a dimeric form of GST P1-1 on western blotting, associated with an increase in the Km(GSH) and a reduction in GST P1-1 specific activity toward 1-chloro-2,4-dinitrobenzene, which reached statistical significance only in H(2)O(2)-treated cells. Our data seem to suggest that H(2)O(2) and etoposide may partly act through a process of partial inactivation of the GST P1-1, possibly involving the "G" site in the process of dimerization, and thus favoring programmed cell death.     

Role of mitogen-activated protein kinases in 4-hydroxy-2-nonenal-induced actin remodeling and barrier function in endothelial cells

In vivo and in vitro studies indicate that 4-hydroxy-2-nonenal (4-HNE), generated by cellular lipid peroxidation or after oxidative stress, affects endothelial permeability and vascular tone. However, the mechanism(s) of 4-HNE-induced endothelial barrier function is not well defined. Here we provide evidence for the first time on the involvement of mitogen-activated protein kinases (MAPKs) in 4-HNE-mediated actin stress fiber formation and barrier function in lung endothelial cells. Treatment of bovine lung microvascular endothelial cells with hydrogen peroxide (H(2)O(2)), as a model oxidant, resulted in accumulation of 4-HNE as evidenced by the formation of 4-HNE-Michael protein adducts. Exposure of cells to 4-HNE, in a dose- and time-dependent manner, decreased endothelial cell permeability measured as transendothelial electrical resistance. The 4-HNE-induced permeability changes were not because of cytotoxicity or endothelial cell apoptosis, which occurred after prolonged treatment and at higher concentrations of 4-HNE. 4-HNE-induced changes in transendothelial electrical resistance were calcium independent, as 4-HNE did not alter intracellular free calcium levels as compared with H(2)O(2) or diperoxovanadate. Stimulation of quiescent cells with 4-HNE (1-100 microm) resulted in phosphorylation of ERK1/2, JNK, and p38 MAPKs, and actin cytoskeleton remodeling. Furthermore, pretreatment of bovine lung microvascular endothelial cells with PD 98059 (25 microm), an inhibitor of MEK1/2, or SP 600125 (25 microm), an inhibitor of JNK, or SB 202190 (25 microm), an inhibitor of p38 MAPK, partially attenuated 4-HNE-mediated barrier function and cytoskeletal remodeling. These results suggest that the activation of ERK, JNK, and p38 MAP kinases is involved in 4-HNE-mediated actin remodeling and endothelial barrier function.     

The rate of cellular hydrogen peroxide removal shows dependency on GSH: mathematical insight into in vivo H2O2 and GPx concentrations

Although its concentration is generally not known, glutathione peroxidase-1 (GPx-1) is a key enzyme in the removal of hydrogen peroxide (H2O2) in biological systems. Extrapolating from kinetic results obtained in vitro using dilute, homogenous buffered solutions, it is generally accepted that the rate of elimination of H2O2 in vivo by GPx is independent of glutathione concentration (GSH). To examine this doctrine, a mathematical analysis of a kinetic model for the removal of H2O2 by GPx was undertaken to determine how the reaction species (H2O2, GSH, and GPx-1) influence the rate of removal of H2O2. Using both the traditional kinetic rate law approximation (classical model) and the generalized kinetic expression, the results show that the rate of removal of H2O2 increases with initial GPx(r), as expected, but is a function of both GPx(r) and GSH when the initial GPx(r) is less than H2O2. This simulation is supported by the biological observations of Li et al. Using genetically altered human glioma cells in in vitro cell culture and in an in vivo tumour model, they inferred that the rate of removal of H2O2 was a direct function of GPx activity x GSH (effective GPx activity). The predicted cellular average GPx(r) and H2O2 for their study are approximately GPx(r) < or =1 microm and H2O2 approximately 5 microm based on available rate constants and an estimation of GSH. It was also found that results from the accepted kinetic rate law approximation significantly deviated from those obtained from the more generalized model in many cases that may be of physiological importance.     

Reversing chemoresistance in cisplatin-resistant human ovarian cancer cells: a role of c-Jun NH2-terminal kinase 1

To investigate the role of activation of c-Jun NH2-terminal kinase 1 (JNK1) in mediating cisplatin-induced apoptosis and the possibility of induction of JNK activity in triggering relation to DNA damage and drug resistance. We investigated the difference of cisplatin-induced activation of JNK pathway and H2O2 alteration between cisplatin-sensitive human ovarian carcinoma cell line A2780 and its resistant variant A2780/DDP. JNK, p-JNK protein, and extracellular H2O2 levels were determined in both A2780 and A2780/DDP cells which were transfected with dominant negative allele of JNK and recombinant JNK1 separately. Both A2780 and A2780/DDP were treated with CDDP, the JNK pathway was activated and a prolonged JNK activation was maintained for at least 12 h in A2780, and only a transient activation (3 h) was detected in A2780/DDP in response to cisplatin treatment. Inhibition of JNK activity by transfection with a dominant negative allele of JNK blocked CDDP-induced apoptosis significantly in A2780 cells. Selective stimulation of the JNK pathway by lipofectamine-mediated delivery of recombinant JNK1 led to activation of c-Jun and decrease of extracellular H2O2, as well as apoptosis sensitization to CDDP in A2780/DDP cells. We concluded that JNK pathway might play an important role in mediating cisplatin-induced apoptosis in A2780 cells, and the duration of JNK activation might be critical in determining whether cells survive or undergo apoptosis. The resistance to CDDP can be reversed through activating c-Jun and decreasing extracellular generation of H2O2 by pcDNA3(FLAG)-JNK1-wt transfection in A2780/DDP cells.     

Structural basis for catalytic differences between alpha class human glutathione transferases hGSTA1-1 and hGSTA2-2 for glutathione conjugation of environmental carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide

The ultimate diol epoxide carcinogens derived from polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BP), are metabolized primarily by glutathione (GSH) conjugation reaction catalyzed by GSH transferases (GSTs). In human liver and probably lung, the alpha class GSTs are likely to be responsible for the majority of this reaction because of their high abundance. The catalytic efficiency for GSH conjugation of the carcinogenic (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide [(+)-anti-BPDE] is more than 5-fold higher for hGSTA1-1 than for hGSTA2-2. Here, we demonstrate that mutation of isoleucine-11 of hGSTA2-2, a residue located in the hydrophobic substrate-binding site (H-site) of the enzyme, to alanine (which is present in the same position in hGSTA1-1) results in about a 7-fold increase in catalytic efficiency for (+)-anti-BPDE-GSH conjugation. Thus, a single amino acid substitution is sufficient to convert hGSTA2-2 to a protein that matches hGSTA1-1 in its catalytic efficiency. The increased catalytic efficiency of hGSTA2/I11A is accompanied by greater enantioselectivity for the carcinogenic (+)-anti-BPDE over (-)-anti-BPDE. Further remodeling of the H-site of hGSTA2-2 to resemble that of hGSTA1-1 (S9F, I11A, F110V, and S215A mutations, SIFS mutant) results in an enzyme whose catalytic efficiency is approximately 13.5-fold higher than that of the wild-type hGSTA2-2, and about 2.5-fold higher than that of the wild-type hGSTA1-1. The increased activity upon mutations can be rationalized by the interactions of the amino acid side chains with the substrate and the orientation of the substrate in the active site, as visualized by molecular modeling. Interestingly, the catalytic efficiency of hGSTA2-2 toward (-)-anti-BPDE was increased to a level close to that of hGSTA1-1 upon F110V, not I11A, mutation. Similar to (+)-anti-BPDE, however, the SIFS mutant was the most efficient enzyme for GSH conjugation of (-)-anti-BPDE.     

Transfection of mGSTA4 in HL-60 cells protects against 4-hydroxynonenal-induced apoptosis by inhibiting JNK-mediated signaling

The mammalian alpha-class glutathione S-transferase (GST) isozymes mGSTA4-4, rGSTA4-4, and hGSTA4-4 are known to utilize 4-hydroxynonenal (4HNE) as a preferred substrate. During the present studies, we have examined the effect of transfecting human myeloid HL-60 cells with mGSTA4, on 4-HNE-induced apoptosis and the associated signaling mechanisms. Results of these studies show that treatment of the wild-type or vector-only-transfected HL-60 cells with 20 microM 4-HNE caused apoptosis within 2 h. The cells transfected with mGSTA4 did not undergo apoptosis under these conditions even after 4 h. In the wild-type and vector-transfected cells, apoptosis was preceded by JNK activation and c-Jun phosphorylation within 30 min, and an increase in AP-1 binding within 2 h of treatment with 20 microM 4-HNE. In mGSTA4-transfected cells, JNK activation and c-Jun phosphorylation were observed after 1 h, and increased AP-1 binding was observed after 8 h under these conditions. In the control cells, 20 microM 4-HNE caused caspase 3 activation and poly(ADP-ribose) polymerase cleavage within 2 h, while in mGSTA4-transfected cells, a lesser degree of these effects was observed even after 8 h. Transfection with mGSTA4 also provided protection to the cells from 4-HNE and doxorubicin cytotoxicity (1.6- and 2.6-fold, respectively). These results show that 4-HNE mediates apoptosis through its effects on JNK and caspase 3, and that 4-HNE metabolizing GST isozyme(s) may be important in the regulation of this pathway of oxidative-stress-induced apoptosis.     

N-acetyl-L-cysteine counteracts oxidative stress and prevents H2O2 induced germ cell apoptosis through down-regulation of caspase-9 and JNK/c-Jun

The mechanism of H(2)O(2) induced oxidative stress leading to male germ cell apoptosis was earlier reported from our laboratory. In the present study, we investigated the mechanisms by which N-acetyl-L-cysteine (NAC, which is highly cell specific with strong antioxidant and anti-genotoxic properties), stimulated cell survival under such conditions. Co-incubation with 5 mM NAC significantly (P<0.001) reduced the germ cell apoptosis induced by 10 μM H(2)O(2). Lipid peroxidation was brought down with significant restoration of activities of antioxidant enzymes, SOD, GST, and catalase. Expression of pro-apoptotic marker, Bax up-regulated following H(2)O(2) exposure, was reversed back to control levels. In contrast, expression of anti-apoptotic Bcl-2 and phospho-Akt revealed a completely opposite trend. While caspase-8 activity remained unaffected, NAC successfully attenuated the increased activities of caspase-3 and -9 in the H(2) O(2) treated cells. Simultaneously, the increased expression of caspase-9, phospho-JNK, and phospho-c-Jun after H(2)O(2) treatment was down-regulated by NAC. The above findings indicate that the mechanism of inhibition of H(2)O(2) induced male germ cell apoptosis by NAC is mediated through regulation of caspase-9 and JNK.     

Oxidative preconditioning and apoptosis in L-cells. Roles of protein kinase B and mitogen-activated protein kinases

Oxidative stress can cause significant cell death by apoptosis. We performed studies in L-cells to explore whether prior exposure to oxidative stress ("oxidative preconditioning") can protect the cell against the apoptotic consequences of subsequent oxidative insults and to establish the mediators in the preconditioning signaling cascade. Cells were preconditioned with three 5-min exposures to H(2)O(2), followed by 10-h recovery and subsequent exposure to 600 microm H(2)O(2) for 10 h. A single 10-h exposure to H(2)O(2) induced substantial apoptotic cell death (approximately 90%), as determined by enzyme-linked immunosorbent assay, TUNEL (terminal deoxyribonucleotide transferase-mediated dUTP nick end labeling), and Annexin V methods, but apoptosis was largely prevented in preconditioned cells. The degree of cytoprotection depended on the strength of preconditioning or H(2)O(2) concentration (20 approximately 600 microm). Transient increases in mitogen-activated protein kinase (MAPK), p38, and JNK/SAPK activities and sustained protein kinase B (Akt) activation, accompanied by drastically reduced caspase 3 activity, were seen after preconditioning. The expression levels of these kinases were unaltered. Inhibitors of p38 (SB203580) and phosphoinositide 3-kinase (PI3K, LY294002) pathways abolished the protection provided by preconditioning. We conclude that oxidative preconditioning protects cells against apoptosis and that this effect involves MAPK and PI3K/Akt pathways. This system may be important in regulating apoptotic cell death in development and disease states.     

GLUT-1 reduces hypoxia-induced apoptosis and JNK pathway activation

Many studies have suggested that enhanced glucose uptake protects cells from hypoxic injury. More recently, it has become clear that hypoxia induces apoptosis as well as necrotic cell death. We have previously shown that hypoxia-induced apoptosis can be prevented by glucose uptake and glycolytic metabolism in cardiac myocytes. To test whether increasing the number of glucose transporters on the plasma membrane of cells could elicit a similar protective response, independent of the levels of extracellular glucose, we overexpressed the facilitative glucose transporter GLUT-1 in a vascular smooth muscle cell line. After 4 h of hypoxia, the percentage of cells that showed morphological changes of apoptosis was 30.5 +/- 2.6% in control cells and only 6.0 +/- 1.1 and 3.9 +/- 0.3% in GLUT-1-overexpressing cells. Similar protection against cell death and apoptosis was seen in GLUT-1-overexpressing cells treated for 6 h with the electron transport inhibitor rotenone. In addition, hypoxia and rotenone stimulated c-Jun-NH(2)-terminal kinase (JNK) activity >10-fold in control cell lines, and this activation was markedly reduced in GLUT-1-overexpressing cell lines. A catalytically inactive mutant of MEKK1, an upstream kinase in the JNK pathway, reduced hypoxia-induced apoptosis by 39%. These findings show that GLUT-1 overexpression prevents hypoxia-induced apoptosis possibly via inhibition of stress-activated protein kinase pathway activation.     

Ceramide increases oxidative damage due to inhibition of catalase by caspase-3-dependent proteolysis in HL-60 cell apoptosis

We investigated through which mechanisms ceramide increased oxidative damage to induce leukemia HL-60 cell apoptosis. When 5 microm N-acetylsphingosine (C(2)-ceramide) or 20 microm H(2)O(2) alone induced little increase of reactive oxygen species (ROS) generation as judged by the 2'-7'-dichlorofluorescin diacetate method, 20 microm H(2)O(2) enhanced oxidative damage as judged by ROS accumulation, and thiobarbituric acid-reactive substance production after pretreatment with 5 microm C(2)-ceramide at least for 12 h. The treatment with a catalase inhibitor, 3-amino-1h-1,2,4-triazole, increased oxidative damage and apoptosis induced by H(2)O(2), and in contrast, purified catalase inhibited the enhancement of oxidative damage by H(2)O(2) in ceramide-pretreated cells, suggesting that the oxidative effect of ceramide is involved in catalase regulation. Indeed, C(2)-ceramide inhibited the activity of immunoprecipitated catalase and decreased the levels of catalase protein in a time-dependent manner. Moreover, acetyl-Asp-Met-Gln-Asp-aldehyde, which dominantly inhibited caspase-3 and blocked the increase of oxidative damage and apoptosis due to C(2)-ceramide-induced catalase depletion at protein and activity levels. In vitro, active and purified caspase-3, but not caspase-6, -8, and -9, inhibited catalase activity and induced the proteolysis of catalase protein whereas these in vitro effects of caspase-3 were blocked by acetyl-Asp-Met-Gln-Asp-aldehyde. Taken together, it is suggested that H(2)O(2) enhances apoptosis in ceramide-pretreated cells, because ceramide increases oxidative damage by inhibition of ROS scavenging ability through caspase-3-dependent proteolysis of catalase.     

Cathepsin D and H2O2 stimulate degradation of thioredoxin-1: implication for endothelial cell apoptosis

Cathepsin D (CatD) is a lysosomal aspartic proteinase and plays an important role in the degradation of proteins and in apoptotic processes induced by oxidative stress, cytokines, and aging. All of these stimuli are potent inducers of endothelial cell apoptosis. Therefore, we investigated the role of CatD in endothelial cell apoptosis and determined the underlying mechanisms. Incubation with 100-500 microm H2O2 for 12 h induced apoptosis in endothelial cells. To determine a role for CatD, we co-incubated endothelial cells with the CatD inhibitor pepstatin A. Pepstatin A as well as genetic knock down of CatD abolished H2O2-induced apoptosis. In contrast, overexpression of CatD wild type but not a catalytically inactive mutant of CatD (CatDD295N) induced apoptosis under basal conditions. To gain insights into the underlying mechanisms, we investigated the effect of CatD on reactive oxygen species (ROS) formation. Indeed, knocking down CatD expression reduced H2O2-induced ROS formation and apoptosis. The major redox regulator in endothelial cells is thioredoxin-1 (Trx), which plays a crucial role in apoptosis inhibition. Thus, we hypothesized that CatD may alter Trx protein levels and thereby promote formation of ROS and apoptosis. Incubation with 100 microm H2O2 for 6 h decreased Trx protein levels, whereas Trx mRNA was not altered. H2O2-induced Trx degradation was inhibited by pepstatin A and genetic knock down of CatD but not by other protease inhibitors. Incubation of unstimulated cell lysates with recombinant CatD significantly reduced Trx protein levels in vitro, which was completely blocked by pepstatin A pre-incubation. Overexpression of CatD reduced Trx protein in cells. Moreover, H2O2 incubation led to a translocation of Trx to the lysosomes prior to the induction of apoptosis. Taken together, CatD induces apoptosis via degradation of Trx protein, which is an essential anti-apoptotic and reactive oxygen species scavenging protein in endothelial cells.     

Hydrogen peroxide-mediated phosphorylation of ERK1/2, Akt/PKB and JNK in cortical neurones: dependence on Ca2+ and PI3-kinase

Primary cortical neurones exposed to an oxidative insult in the form of hydrogen peroxide (H(2)O(2)) for 30 min showed a concentration-dependent increase in oxidative stress followed by a delayed NMDA receptor-dependent cell death measured 24 h later. Extracellular signal-regulated protein kinase (ERK1/2), c-jun N-terminal kinase (JNK) and the kinase Akt/PKB may regulate neuronal viability in response to oxidative insults. Using phospho-specific antibodies, a 15-min stimulation of neurones with H(2)O(2) (100 microm - 1 mm) produced a concentration-dependent phosphorylation of ERK1/2 and Akt/PKB that was partly dependent on extracellular Ca(2+) and phosphatidylinositol 3-kinase (PI3-K). Higher concentrations of H(2)O(2) (1 mm) also stimulated a phosphorylation of JNK which was totally dependent on extracellular Ca(2+) but not PI3-K. H(2)O(2)-induced phosphorylation of ERK1/2, Akt/PKB or JNK were unaffected by the NMDA channel blocker MK801. Blocking ERK1/2 activation with the upstream inhibitor U0126 (10 microm) enhanced H(2)O(2)-induced (100-300 microm range) neurotoxicity and inhibited H(2)O(2)-mediated phosphorylation of the cyclic AMP regulatory binding protein (CREB), suggesting that ERK1/2 signals to survival under these conditions. At higher concentrations (mm), H(2)O(2)-stimulated a phosphorylation of c-jun. It is likely, therefore, that subjecting neurones to moderate oxidative-stress recruits pro-survival signals to CREB but during severe oxidative stress pro-death signals through JNK and c-jun are dominant.     

Phosphorylation of proteins and apoptosis induced by c-Jun N-terminal kinase1 activation in rat cardiomyocytes by H(2)O(2) stimulation

Cytokines and various cellular stresses are known to activate c-Jun N-terminal kinase-1 (JNK1), which is involved in physiological function. Here, we investigate the activation of JNK1 by oxidative stress in H9c2 cells derived from rat cardiomyocytes. H(2)O(2) (100 microM) significantly induces the tyrosine phosphorylation of JNK1 with a peak 25 min after the stimulation. The amount of JNK1 protein remains almost constant during stimulation. Immunocytochemical observation shows that JNK1 staining in the nucleus is enhanced after H(2)O(2) stimulation. To clarify the physiological role of JNK1 activation under these conditions, we transfected antisense JNK1 DNA into H9c2 cells. The antisense DNA (2 microM) inhibits JNK1 expression by 80% as compared with expression in the presence of the sense DNA, and significantly blocks H(2)O(2)-induced cell death. Consistent with the decrease in cell number, we detected condensation of the nuclei, a hallmark of apoptosis, 3 h after H(2)O(2) stimulation in the presence of the sense DNA for JNK1. The antisense DNA of JNK1 inhibits the condensation of nuclei by H(2)O(2). Under these conditions, the H(2)O(2)-induced phosphorylation of proteins with molecular masses of 55, 72, and 78 kDa is blocked by treatment with the antisense DNA for JNK1 as compared with the sense DNA for JNK1. These findings suggest that JNK1 induces apoptotic cell death in response to H(2)O(2), and that the cell death may be involved in the phosphorylations of 55, 72, and 78 kDa proteins induced by JNK1 activation.