Phospholipase C-dependent hydrolysis of phosphatidylcholine hydroperoxides to diacylglycerol hydroperoxides and its reduction by phospholipid hydroperoxide glutathione peroxidase

We have shown that 1,2-diacylglycerol hydroperoxides activate protein kinase C (PKC) as efficiently as does phorbol ester [Takekoshi S, Kambayashi Y, Nagata H, Takagi T, Yamamoto Y, Watanabe K. Activation of protein kinase C by oxidized diacylglycerol. Biochem Biophys Res Commun 1995; 217: 654-660]. 1,2-Diacylglycerol hydroperoxides also stimulate human neutrophils to release superoxide whereas their hydroxides do not [Yamamoto Y, Kambayashi Y, Ito T, Watanabe K, Nakano M. 1,2-Diacylglycerol hydroperoxides induce the generation and release of superoxide anion from human polymorphonuclear leukocytes. FEBS Lett 1997; 412: 461-464]. One of the proposed mechanisms for the formation of 1,2-diacylglycerol hydroperoxides is the hydrolysis of phosphatidylcholine hydroperoxides by phospholipase C (PLC). To confirm this hypothesis, we incubated 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) liposomes containing PLPC hydroperoxides (PLPC-OOH) with Bacillus cereus PLC and found 1-palmitoyl-2-linoleoylglycerol (PLG) and its hydroperoxide (PLG-OOH) were produced. PLC hydrolyzed the two substrates without preference, as the yields of PLG and PLG-OOH were the same even though cholesterol was incorporated into liposomes to increase bilayer integrity. Phospholipid hydroperoxide glutathione peroxidase (PHGPX) reduced PLG-OOH to its hydroxide in the presence of glutathione while the conventional cytosolic glutathione peroxidase did not. These data suggest that PLC hydrolyzes oxidized biomembranes to give 1,2-diacylglycerol hydroperoxides for PKC stimulation but PHGPX may prevent neutrophil stimulation by reducing 1,2-diacylglycerol hydroperoxides to their hydroxides.     

Phospholipase C-dependent hydrolysis of phosphatidylcholine hydroperoxides to diacylglycerol hydroperoxides and its reduction by phospholipid hydroperoxide glutathione peroxidase

Abstract

We have shown that 1,2-diacylglycerol hydroperoxides activate protein kinase C (PKC) as efficiently as does phorbol ester [Takekoshi S, Kambayashi Y, Nagata H, Takagi T, Yamamoto Y, Watanabe K. Activation of protein kinase C by oxidized diacylglycerol. Biochem Biophys Res Commun 1995; 217: 654-660]. 1,2-Diacylglycerol hydroperoxides also stimulate human neutrophils to release superoxide whereas their hydroxides do not [Yamamoto Y, Kambayashi Y, Ito T, Watanabe K, Nakano M. 1,2-Diacylglycerol hydroperoxides induce the generation and release of superoxide anion from human polymorphonuclear leukocytes. FEBS Lett 1997; 412: 461-464]. One of the proposed mechanisms for the formation of 1,2-diacylglycerol hydroperoxides is the hydrolysis of phosphatidylcholine hydroperoxides by phospholipase C (PLC). To confirm this hypothesis, we incubated 1-palmitoyl-2-linoleoyl-phosphatidylcholine (PLPC) liposomes containing PLPC hydroperoxides (PLPC-OOH) with Bacillus cereus PLC and found 1-palmitoyl-2-linoleoylglycerol (PLG) and its hydroperoxide (PLG-OOH) were produced. PLC hydrolyzed the two substrates without preference, as the yields of PLG and PLG-OOH were the same even though cholesterol was incorporated into liposomes to increase bilayer integrity. Phospholipid hydroperoxide glutathione peroxidase (PHGPX) reduced PLG-OOH to its hydroxide in the presence of glutathione while the conventional cytosolic glutathione peroxidase did not. These data suggest that PLC hydrolyzes oxidized biomembranes to give 1,2-diacylglycerol hydroperoxides for PKC stimulation but PHGPX may prevent neutrophil stimulation by reducing 1,2-diacylglycerol hydroperoxides to their hydroxides.     

Embryonic expression profile of phospholipid hydroperoxide glutathione peroxidase

The selenoenzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) is indispensable for murine embryonic development; yet, the cellular mechanisms leading to embryonic death around gastrulation are still unclear. To investigate PHGPx expression patterns during embryogenesis, we performed a detailed analysis that revealed a complex expression profile. Up to embryonic day 9.5, PHGPx was ubiquitously expressed, which was, albeit to a lower extent, maintained throughout later stages of embryogenesis. Notably, strong expression was frequently observed in epithelial tissue. A transient increase in PHGPx expression was detected in developing tissues, suggesting a crucial role for PHGPx in proliferation and differentiation. By semi-quantitative RT-PCR analysis we observed that the cytosolic form of PHGPx was present in embryonic and somatic tissues whereas the mitochondrial and nuclear forms were detectable only in testicular tissue. This strongly suggests that it is the cytosolic form of PHGPx that is indispensable for embryonic development.     

谷胱甘肽磷脂氢过氧化物酶研究进展

谷胱甘肽磷脂氢过氧化物酶(PHGPx)是生物体内一种重要的抗氧化酶。它是一种硒依赖性蛋白,在谷胱甘肽(GSH)的参与下能特异性地还原磷脂氢过氧化物(PLOOH)和胆固醇氢过氧化物(ChOOH),从而保护生物膜免受过氧化损伤。它还是核酸等生物大分子的重要保护剂,并且在细胞凋亡调控中发挥作用。     

Testis glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase activities in aminoguanidine-treated diabetic rats

Severe steroidogenic and spermatogenic alterations are reported in association with diabetic manifestations in humans and experimental animals. This study was planned to determine whether oxidative stress is involved in diabetes-induced alterations in the testes. Diabetes was induced in male rats by injection of 50 mg/kg of streptozotocin (STZ). Ten weeks after injection of STZ, levels of selenium and activities of selenium dependent-glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) were measured in rat testis. Lipid and protein oxidations were evaluated as measurements of testis malondialdehyde (MDA) and protein carbonyl levels, respectively. Testis sulfydryl (SH) levels were also determined. The control levels of GPx and PHGPx activities were found to be 46.5 +/- 6.2 and 108.8 +/- 19.8 nmol GSH/mg protein/min, respectively. Diabetes caused an increase in testis GPx (65.0 +/- 21.1) and PHGPx (155.9 +/- 43.1) activities but did not affect the levels of selenium or SH. However, the testis MDA and protein carbonyl levels as markers of lipid and protein oxidation, respectively, did not increase in the diabetic group. Aminoguanidine (AG) treatment of diabetic rats returned the testis PHGPx activity (136.5 +/- 24.9) to the control level but did not change the value of GPx activity (69.2 +/- 17.4) compared with diabetic group. MDA and protein carbonyl levels in testis were not affected by AG treatment of diabetic rats, but interestingly AG caused SH levels to increase. The results indicate that reactive oxygen radicals were not involved in possible testicular complications of diabetes because diabetes-induced activations of GPx and PHGPx provided protection against oxidative stress, which was reported to be related to some diabetic complications.     

Physiological role of phospholipid hydroperoxide glutathione peroxidase in mammals

The redox enzyme phospholipid hydroperoxide glutathione peroxidase (PHGPx) has emerged as one of the most significant selenoenzymes in mammals, corroborated by early embryonic lethality of PHGPx null mice. PHGPx is one of five selenium-dependent glutathione peroxidases and the second glutathione peroxidase to be discovered in 1982. PHGPx has a particular position within this family owing to its peculiar structural and catalytic properties, its multifaceted roles during male gametogenesis, and its necessity for early mouse development. Interestingly, mice devoid of endogenous glutathione die at the same embryonic stage as PHGPx-deficient mice compatible with the hypothesis that a similar phenotype of embryonic lethality may be provoked by PHGPx deficiency and lack of its reducing substrate glutathione. Various gain- and loss-of-function approaches in mice have provided some insights into the physiological functions of PHGPx. These include a protective role for PHGPx in response to irradiation, increased resistance of transgenic PHGPx mice to toxin-induced liver damage, a putative role in various steps of embryogenesis, and a contribution to sperm chromatin condensation. The expression of three forms of PHGPx and early embryonic lethality call for more specific studies, such as tissue-specific disruption of PHGPx, to precisely understand the contribution of PHGPx to mammalian physiology and under pathological conditions.     

Protective effects of glutathione against lipid peroxidation in chronically iron-loaded mice

To elucidate the protective effects of glutathione against iron-induced peroxidative injury, changes in the hepatic glutathione metabolism were studied in chronically iron-loaded mice. When the diets of the mice were supplemented with carbonyl iron, iron deposition occurred primarily in the parenchymal cells of the liver. In addition, expiratory ethane production was elevated, suggesting an enhancement in lipid peroxidation. In iron-loaded mice, the total hepatic glutathione contents were higher (6.21 +/- 0.53 mumol/g wet wt.) than in control mice (4.61 +/- 0.31 mumol/g wet wt.), primarily due to an increase in the reduced glutathione contents. The value of oxidized glutathione was also higher (98.5 +/- 8.1 nmol/g wet wt.) than in the controls (60.8 +/- 9.5 nmol/g wet wt.), and the ratio of oxidized glutathione to total glutathione increased. The excretion rate of glutathione from the hepatocytes in iron-loaded mice also increased. These observations suggest that chronic iron-loading of mice stimulates lipid peroxidation and oxidation of glutathione and that peroxidized molecules may be catabolized using reduced glutathione.     

Involvement of mitochondrial phospholipid hydroperoxide glutathione peroxidase as an antiapoptotic factor

Although reactive oxygen species (ROS) such as superoxide and hydroperoxide are known to induce apoptotic cell death, little is known as to the apoptotic death signaling of mitochondrial ROS. Recent evidence has suggested that antioxidant enzymes in mitochondria may be responsible for the regulation of cytochrome c release and apoptotic cell death. This paper examines the current state of knowledge regarding the role of mitochondrial antioxidant enzymes, especially phospholipid hydroperoxide glutathione peroxidase. A model for the release of cytochrome c by lipid hydroperoxide has also been proposed.     

The role of phospholipid hydroperoxide glutathione peroxidase isoforms in murine embryogenesis

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a selenocysteine-containing enzyme, and three different isoforms (cytosolic, mitochondrial, and nuclear) originate from the GPx4 gene. Homozygous GPx4-deficient mice die in utero at midgestation, since they fail to initiate gastrulation and do not develop embryonic cavities. To investigate the biological basis for embryonic lethality, we first explored expression of the GPx4 in adult murine brain and found expression of the protein in cerebral neurons. Next, we profiled mRNA expression during the time course of embryogenesis (embryonic days 6.5-17.5 (E6.5-17.5)) and detected mitochondrial and cytosolic mRNA species at high concentrations. In contrast, the nuclear isoform was only expressed in small amounts. Cytosolic GPx4 mRNA was present at constant levels (about 100 copies per 1000 copies of glyceraldehyde-3-phosphate dehydrogenase mRNA), whereas nuclear and mitochondrial isoforms were down-regulated between E14.5 and E17.5. In situ hybridization indicated expression of GPx4 isoforms in all developing germ layers during gastrulation and in the somite stage in the developing central nervous system and in the heart. When we silenced expression of GPx4 isoforms during in vitro embryogenesis using short interfering RNA technology, we observed that knockdown of mitochondrial GPx4 strongly impaired segmentation of rhombomeres 5 and 6 during hindbrain development and induced cerebral apoptosis. In contrast, silencing expression of the nuclear isoform led to retardations in atrium formation. Taken together, our data indicate specific expression of GPx4 isoforms in embryonic brain and heart and strongly suggest a role of this enzyme in organogenesis. These findings may explain in part intrauterine lethality of GPx4 knock-out mice.     

Prevention of geranylgeranoic acid-induced apoptosis by phospholipid hydroperoxide glutathione peroxidase gene

Micromolar concentrations (0.5 approximately 5 microM) of all-trans geranylgeranoic acid (GGA) induced cell death in a guinea pig cell line, 104C1, whereas under the same conditions GGA was unable to kill 104C1/O4C, a clone established from 104C1 cells by transfection of them with the human phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene. GGA (5 microM) induced a loss of the mitochondrial inner membrane potential (DeltaPsim) in 104C1 cells in 2 h, and their apoptotic cell death became evident in 6 h. On the other hand, 104C1/O4C cells were resistant to loss of DeltaPsim and showed intact morphology until at least 24 h after addition of 10 microM GGA. Dihydroethidine, superoxide-sensitive probe, was immediately oxidized 15 min after addition of GGA in both 104C1 and 104C1/O4C cells. The peroxide-sensitive probe 2',7'-dichlorofluorescin diacetate (H2-DCF-DA) was strongly oxidized in 104C1 cells 4 h after the addition of 2.5 microM GGA, but not in 104C1/O4C cells even in the presence of 10 microM GGA. The present results suggest that GGA induced a hyper-production of superoxide and subsequently peroxides, which in turn may have led to dissipation of the DeltaPsim and final apoptotic cell death in 104C1 cells.     

Mitochondrial phospholipid hydroperoxide glutathione peroxidase suppresses apoptosis mediated by a mitochondrial death pathway.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a key enzyme in the protection of biomembranes exposed to oxidative stress. We investigated the role of mitochondrial PHGPx in apoptosis using RBL2H3 cells that overexpressed mitochondrial PHGPx (M15 cells), cells that overexpressed non-mitochondrial PHGPx (L9 cells), and control cells (S1 cells). The morphological changes and fragmentation of DNA associated with apoptosis occurred within 15 h in S1 and L9 cells upon exposure of cells to 2-deoxyglucose (2DG). The release of cytochrome c from mitochondria was observed in S1 cells after 4 h and was followed by the activation of caspase-3 within 6 h. Overexpression of mitochondrial PHGPx prevented the release of cytochrome c, the activation of caspase-3, and apoptosis, but non-mitochondrial PHGPx lacked the ability to prevent the induction of apoptosis by 2DG. An ability to protect cells from 2DG-induced apoptosis was abolished when the PHGPx activity of M15 cells was inhibited by diethylmalate, indicating that the resistance of M15 cells to apoptosis was indeed due to the overexpression of PHGPx in the mitochondria. The expression of members of the Bcl-2 family of proteins, such as Bcl-2, Bcl-xL, Bax, and Bad, was unchanged by the overexpression of PHGPx in cells. The levels of hydroperoxides, including hydrogen and lipid peroxide, in mitochondria isolated from S1 and L9 cells were significantly increased after the exposure to 2DG for 2 h, while the level of hydroperoxide in mitochondria isolated from M15 cells was lower than that in S1 and L9 cells. M15 cells were also resistant to apoptosis induced by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, but not to apoptosis induced by Fas-specific antibodies, which induces apoptosis via a pathway distinct from the pathway initiated by 2DG. Our results suggest that hydroperoxide, produced in mitochondria, is a major factor in apoptosis and that mitochondrial PHGPx might play a critical role as an anti-apoptotic agent in mitochondrial death pathways.     

Photooxidation of cell membranes in the presence of hematoporphyrin derivative: reactivity of phospholipid and cholesterol hydroperoxides with glutathione peroxidase

The susceptibility of photodynamically-generated lipid hydroperoxides to reductive inactivation by glutathione peroxidase (GPX) has been investigated, using hematoporphyrin derivative as a photosensitizing agent and the human erythrocyte ghost as a target membrane. Photoperoxidized ghosts were reactive in a glutathione peroxidase/reductase (GPX/GRD)-coupled assay only after phospholipid hydrolysis by phospholipase A2 (PLA2). However, enzymatically determined lipid hydroperoxide values were consistently approx. 40% lower than iodometrically determined values throughout the course of photooxidation. Moreover, when irradiated ghosts were analyzed iodometrically during PLA2/GSH/GPX treatment, a residual 30-40% of non-reactive lipid hydroperoxide was observed. The possibility that cholesterol product(s) account for the non-reactive lipid hydroperoxide was examined by tracking cholesterol hydroperoxides in [14C]cholesterol-labeled ghosts. The sum of cholesterol hydroperoxides and GPX/GRD-detectable lipid hydroperoxides was found to agree closely with iodometrically determined lipid hydroperoxide throughout the course of irradiation. Thin-layer chromatography of total lipid extracts indicated that cholesterol hydroperoxide was unaffected by PLA2/GSH/GPX treatment, whereas most of the phospholipid peroxides were completely hydrolyzed and the released fatty acid peroxides were reduced to alcohols. It appears, therefore, that the GPX-resistant lipid hydroperoxides in photooxidized ghosts were derived primarily from cholesterol. Ascorbate plus Fe3+ produced a burst of free-radical lipid peroxidation in photooxidized, PLA2-treated ghosts. As expected for fatty acid hydroperoxide inactivation, the lipid peroxidation was inhibited by GSH/GPX, but only partially so, suggesting that cholesterol hydroperoxide-derived radicals play a major role in the reaction.     

The influence of phospholipid polar head on the lipid hydroperoxide dependent initiation of lipid peroxidation.

In a buffer (Mes) and at a pH (6.5) where Fe2+ is very stable, we have studied the peroxidation of liposomes catalyzed by FeCl2. The liposomes studied, prepared by sonolysis, contained either phosphatidylcholine or 1:1 molar ratio of phosphatidylcholine and phosphatidic acid. The presence of the negatively charged phospholipid causes: 1) rapid Fe2+ oxidation and oxygen consumption; 2) increased generation of lipid hydroperoxides; 3) decreased generation of thiobarbituric acid-reactive materials; 4) very low inhibition of Fe2+ oxidation and lipid hydroperoxide generation by BHT; 5) inhibition of the termination phase of lipid peroxidation at high FeCl2 concentrations. A hypothesis is proposed to explain the results obtained.     

Enzymatic reduction of phospholipid and cholesterol hydroperoxides in artificial bilayers and lipoproteins

Lipid hydroperoxides (LOOHs) in various lipid assemblies are shown to be efficiently reduced and deactivated by phospholipid hydroperoxide glutathione peroxidase (PHGPX), the second selenoperoxidase to be identified and characterized. Coupled spectrophotometric analyses in the presence of NADPH, glutathione (GSH), glutathione reductase and Triton X-100 indicated that photochemically generated LOOHs in small unilamellar liposomes are substrates for PHGPX, but not for the classical glutathione peroxidase (GPX). PHGPX was found to be reactive with cholesterol hydroperoxides as well as phospholipid hydroperoxides. Kinetic iodometric analyses during GSH/PHGPX treatment of photoperoxidized liposomes indicated a rapid decay of total LOOH to a residual level of 35-40%; addition of Triton X-100 allowed the reaction to go to completion. The non-reactive LOOHs in intact liposomes were shown to be inaccessible groups on the inner membrane face. In the presence of iron and ascorbate, photoperoxidized liposomes underwent a burst of thiobarbituric acid-detectable lipid peroxidation which could be inhibited by prior GSH/PHGPX treatment, but not by GSH/GPX treatment. Additional experiments indicated that hydroperoxides of phosphatidylcholine, cholesterol and cholesteryl esters in low-density lipoprotein are also good substrates for PHGPX. An important role of PHGPX in cellular detoxification of a wide variety of LOOHs in membranes and internalized lipoproteins is suggested from these findings.     

Inhibitory effect of phospholipid hydroperoxide glutathione peroxidase on the activity of lipoxygenases and cyclooxygenases

The partially purified phospholipid hydroperoxide glutathione peroxidase (PHGPx) from A431 cells was used to systematically compare the inhibitory effect on the enzyme activity of various lipoxygenases and cyclooxygenases. Under the standard assay system, platelet 12-lipoxygenase, 15-lipoxygenase, and cyclooxygenase-2 were the most sensitive to the inhibition by PHGPx. 5-Lipoxygenase and cyclooxygenase-1 were less sensitive to the inhibition by PHGPx than platelet 12-lipoxygenase and cyclooxygenase-2, respectively, and the difference was approximately 10-fold. Reduction of 12(S)-hydroperoxyeicosatetraenoic acid to 12(S)-hydroxyeicosatetraenoic acid by PHGPx was observed in the presence of glutathione (GSH), and the inhibitory effect of PHGPx on 12-lipoxygenase-catalyzed arachidonate metabolism was reversed by the addition of exogenous lipid hydroperoxide. The results indicate that PHGPx directly reduced lipid hydroperoxides and then down-regulated the activity of arachidonate oxygenases. Moreover, a high-level expression of PHGPx mRNA and its 12-lipoxygenase-inhibitory activity was observed in cancer cells and endothelial cells, and these results suggest that PHGPx may play a significant role in the regulation of reactive oxygen species formation in these cells.     

Molecular cloning and functional expression of nucleolar phospholipid hydroperoxide glutathione peroxidase in mammalian cells

We cloned a full-length cDNA for phospholipid hydroperoxide glutathione peroxidase (PHGPx) including exon Ib from rat and mouse testis. The nuclear signal sequence of the N terminal of rat nuclear PHGPx possessed a different sequence from that previously reported for rat sperm nuclei GPx (SnGPx). Expression of this PHGPx-YFP (yellow fluorescent protein) fusion protein including a novel nuclear signal sequence was exclusively localized in nucleolus; although YFPs fused with only a novel nuclear signal sequence were distributed in the whole nucleus, indicating that preferential translocation of nucleolar PHGPx into nucleoli was required for the nuclear signal sequence and internal sequence of PHGPx. Low level expression of nucleolar PHGPx was detected in several tissues, but the expression of nucleolar PHGPx was extensively high in testis. Immunohistochemical analysis with anti-nucleolar PHGPx indicated that expression of nucleolar PHGPx was observed in the nucleoli in the spermatogonia, spermatocyte, and spermatid. Overexpression of 34kDa nucleolar PHGPx in RBL2H3 cells significantly suppressed cell death induced by actinomycin D and doxorubicin that induced damage in the nucleolus. These results indicated that nucleolar PHGPx plays an important role in prevention of nucleolus from damage in mammalian cells.