Biosynthesis of hepoxilins: evidence for the presence of a hepoxilin synthase activity in rat insulinoma cells

The 12(S)-lipoxygenase (12-LOX) pathway of arachidonic acid (AA) metabolism after dioxygenation to 12(S)-hydroperoxy-eicosatetraenoic acid is bifurcated in a reduction route to formation of 12(S)-hydroxy-eicosatetraenoic acid (12-HpETE) and an isomerization route to formation of hepoxilins. Interestingly, we found that the rat insulinoma RINm5F cells, which are devoid of cytoplasmic glutathione peroxidase (cGPx)/phospholipid hydroperoxide glutathione peroxidase (PHGPx), produce solely hepoxilin A(3) (HXA(3)). Since HXA(3) synthesis was abolished in heat-denatured or cGPx- or PHGPx-transfected cells, it was tempting to speculate that a HXA(3) synthase activity regulated by cGPx/PHGPx is present. To confirm this assumption we incubated AA with HeLa cells overexpressing the rat leukocyte-type 12-LOX. Neither HXA(3) nor 12(S)-HETE were detected due to abundance of cGPx/PHGPx. But, pretreatment of transfected cells with diethyl maleate, an inhibitor of glutathione and PHGPx, restored HXA(3) synthase and 12-LOX activities. Thus, we conclude, that cells containing rat leukocyte-type 12-LOX also possess an intrinsic HXA(3) synthase activity, which is activated by inhibition of cGPx/PHGPx. In normal cells HXA(3) is down-regulated by cGPx/PHGPx, but, it is persistently activated in oxidatively stressed cells deficient in cGPx/PHGPx, such as RINm5F.     

Glutathione peroxidases and redox-regulated transcription factors

Analysis of the selenoproteome identified five glutathione peroxidases (GPxs) in mammals: cytosolic GPx (cGPx, GPx1), phospholipid hydroperoxide GPx (PHGPX, GPx4), plasma GPx (pGPX, GPx3), gastrointestinal GPx (GI-GPx, GPx2) and, in humans, GPx6, which is restricted to the olfactory system. GPxs reduce hydroperoxides to the corresponding alcohols by means of glutathione (GSH). They have long been considered to only act as antioxidant enzymes. Increasing evidence, however, suggests that nature has not created redundant GPxs just to detoxify hydroperoxides. cGPx clearly acts as an antioxidant, as convincingly demonstrated in GPx1-knockout mice. PHGPx specifically interferes with NF-kappaB activation by interleukin-1, reduces leukotriene and prostanoid biosynthesis, prevents COX-2 expression, and is indispensable for sperm maturation and embryogenesis. GI-GPx, which is not exclusively expressed in the gastrointestinal system, is upregulated in colon and skin cancers and in certain cultured cancer cells. GI-GPx is a target for Nrf2, and thus is part of the adaptive response by itself, while PHGPx might prevent cancer by interfering with inflammatory pathways. In conclusion, cGPx, PHGPx and GI-GPx have distinct roles, particularly in cellular defence mechanisms. Redox sensing and redox regulation of metabolic events have become attractive paradigms to unravel the specific and in part still enigmatic roles of GPxs.     

The role of selenium peroxidases in the protection against oxidative damage of membranes

The present review deals with the chemical properties of selenium in relation to its antioxidant properties and its reactivity in biological systems. The interaction of selenite with thiols and glutathione and the reactivity of selenocompounds with hydroperoxides are described. After a short survey on distribution, metabolism and organification of selenium, the role of this element as a component of the two seleno-dependent glutathione peroxidases is described. The main features of glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase are also reviewed. Both enzymes reduce different hydroperoxides to the corresponding alcohols and the major difference is the reduction of lipid hydroperoxides in membrane matrix catalyzed only by the phospholipid hydroperoxide glutathione peroxidase. However, in spite of the different specificity for the peroxidic substrates, the kinetic mechanism of both glutathione peroxidase and phospholipid hydroperoxide glutathione peroxidase seems identical and proceeds through a tert-uni ping pong mechanism. In the reaction cycle, indeed, as supported by the kinetic data, the oxidation of the ionized selenol by the hydroperoxide yields a selenenic acid that in turn is reduced back by two reactions with reduced glutathione. Special emphasis has been given to the role of selenium-dependent glutathione peroxidases in the prevention of membrane lipid peroxidation. While glutathione peroxidase is able to reduce hydrogen peroxide and other hydroperoxides possibly present in the soluble compartment of the cell, this enzyme fails to inhibit microsomal lipid peroxidation induced by NADPH or ascorbate and iron complexes. On the other hand, phospholipid hydroperoxide glutathione peroxidase, by reducing the phospholipid hydroperoxides in the membranes, actively prevents lipid peroxidation, provided a normal content of vitamin E is present in the membranes. In fact, by preventing the free radical generation from lipid hydroperoxides, phospholipid hydroperoxide glutathione peroxidase decreases the vitamin E requirement necessary to inhibit lipid peroxidation. Finally, the possible regulatory role of the selenoperoxidases on the arachidonic acid cascade enzymes (cyclooxygenase and lipoxygenase) is discussed.     

Purification of a cytosolic enzyme from human liver with phospholipid hydroperoxide glutathione peroxidase activity

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is a selenoprotein which inhibits peroxidation ofmicrosomes. The human enzyme, which may play an important role in protecting the cell from oxidative damage, has not been purified or characterized. PHGPx was isolated from human liver using ammonium sulphate fractionation, affinity chromatography on bromosulphophthalein-glutathione-agarose, gel filtration on Sephadex G-50, anion exchange chromatography on Mono Q resin and high resolution gel filtration on Superdex 75. The protein was purified about 112,000-fold, and 12 μg, was obtained from 140 g of human liver with a 9% yield. PHGPx was active on hydrogen peroxide, cumene hydroperoxide, linoleic acid hydroperoxide and phosphatidylcholine hydroperoxide. The molecular weight, as estimated from non-denaturing gel filtration, was 16,100. The turnover number (37°C, pH 7.6) on (β-(13-hydroperoxy-cis-9, trans-11-octadecadienoyl)-γ-palmitoyl)-l-α-phosphatidylcholine was 91 mol mo⁻¹ s⁻¹. As reported for pig PHGPx, activity of the enzyme from human liver on cumene hydroperoxide and on linoleic acid hydroperoxide was inhibited by deoxycholate. In the presence of glutathione, the enzyme was a potent inhibitor of ascorbate/Fe induced lipid peroxidation in microsomes derived from human B lymphoblastic AHH-1 TK ± CHol cells but not from human liver microsomes. Human cell line microsomes contained no detectable PHGPx activity. However, microsomes prepared from human liver contained 0.009 U/mg of endogenous PHGPx activity, which is 4–5 times the activity required for maximum inhibition of lipid peroxidation when pure PHGPx was added back to human lymphoblastic cell microsomes. PHGPx from human liver exhibits similar properties to previously described enzymes with PHGPx activity isolated from pig and rat tissues, but does not inhibit peroxidation of human liver microsomes owing to a high level of PHGPx activity already present in these microsomes.     

Inhibition of arachidonate metabolism in human epidermoid carcinoma a431 cells overexpressing phospholipid hydroperoxide glutathione peroxidase

Phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenium-dependent glutathione peroxidase, can interact with lipophilic substrates, including phospholipid hydroperoxides, fatty acid hydroperoxides and cholesterol hydroperoxides, and can reduce them to hydroxide compounds. It also seems to be a major regulator of lipid oxygenation in human epidermoid carcinoma A431 cells. In order to study the functional role of PHGPx in the regulation of 12-lipoxygenase and cyclooxygenase, cDNA of PHGPx was inserted into pcDNA3.1/His, and a plasmid designated as S4 with the His-tag sequence inserted between PHGPx and its 3'-untranslated region was constructed. A number of stable transfectants of A431 cells that could express the tag-PHGPx were generated using plasmid S4. Using an intact cell assay system, the metabolism of arachidonic acid to prostaglandin E(2) significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line. If the intact cell assay was carried out in the presence of 13-hydroperoxyoctadecadienoic acid as a stimulator of lipid peroxidation, formation of 12-hydroxyeicosatetraenoic acid from arachidonic acid also significantly decreased in stable transfectants of overexpressing PHGPx compared to that in a vector control cell line, indicating that PHGPx could downregulate the 12-lipoxygenase activity in cells. These results support the hypothesis that PHGPx plays a pivotal role in the regulation of arachidonate metabolism in A431 cells.     

Distribution of glutathione peroxidases glutathione reductase in rat brain mitochondria

The distribution of glutathione reductase (GR), glutathione peroxidase (GPx) and phospholipid hydroperoxide glutathione peroxidase (PHGPx) in isolated rat brain mitochondria was investigated. using a fractionation procedure for the separation of inner and outer membranes, contact sites between the two membranes and a soluble fraction mainly originating from the mitochondrial matrix. The data indicate that GR and GPx are concentrated in the soluble fraction, with a minor portion of the two enzymes being associated with the contact sites. PHGPx is localized largely in the inner membrane. The possible functional significance of these findings is discussed.     

Saccharomyces cerevisiae expresses three phospholipid hydroperoxide glutathione peroxidases.

The GPX1, GPX2, and GPX3 genes of Saccharomyces cerevisiae have been reported previously to encode glutathione peroxidases (GPxs). We re-examined the sequence alignments of these proteins with GPxs from higher eukaryotes. Sequence identities, particularly with phospholipid hydroperoxide glutathione peroxidases (PHGPxs), were enhanced markedly by introduction to the yeast sequences of gaps that are characteristic of PHGPxs. PHGPx-like activity was detectable in extracts from wild-type S. cerevisiae and was diminished in extracts from gpx1 Delta, gpx2 Delta, and gpx3 Delta deletion mutants; PHGPx activity was almost absent in a gpx1 Delta/gpx2 Delta/gpx3 Delta triple mutant. Studies with cloned GPX1, GPX2, and GPX3 expressed heterologously in Escherichia coli confirmed that these genes encode proteins with PHGPx activity. An S. cerevisiae gpx1 Delta/gpx2 Delta/gpx3 Delta mutant was defective for growth in medium supplemented with the oxidation-sensitive polyunsaturated fatty acid linolenate (18:3). This sensitivity to 18:3 was more marked than sensitivity to H(2)O(2). Unlike H(2)O(2) toxicity, delayed toxicity of 18:3 toward gpx1 Delta/gpx2 Delta/gpx3 Delta cells was correlated with the gradual incorporation of 18:3 into S. cerevisiae membrane lipids and was suppressible with alpha-tocopherol, an inhibitor of lipid peroxidation. The results show that the GPX genes of S. cerevisiae, previously reported to encode GPxs, encode PHGPxs (PHGPx1, PHGPx2, and PHGPx3) and that these enzymes protect yeast against phospholipid hydroperoxides as well as nonphospholipid peroxides during oxidative stress. This is the first report of an organism that expresses PHGPx from more than one gene and produces PHGPx in the absence of a GPx.     

Tissue-specific functions of individual glutathione peroxidases

The family of glutathione peroxidases comprises four distinct mammalian selenoproteins. The classical enzyme (cGPx) is ubiquitously distributed. According to animal, cell culture and inverse genetic studies, its primary function is to counteract oxidative attack. It is dispensible in unstressed animals, and accordingly ranks low in the hierarchy of glutathione peroxidases. The gastrointestinal isoenzyme (GI-GPx) is most related to cGPx and is exclusively expressed in the gastrointestinal tract. It might provide a barrier against hydroperoxides derived from the diet or from metabolism of ingested xenobiotics. The extreme stability in selenium deficiency ranks this glutathione peroxidase highest in the hierarchy of selenoproteins and points to a more vital function than that of cGPx. Plasma GPx (pGPx) behaves similar to cGPx in selenium deficiency. It is directed to extracellular compartments and is expressed in various tissues in contact with body fluids, e.g., kidney, ciliary body, and maternal/fetal interfaces. It has to be rated as an efficient extracellular antioxidant device, though with low capacity because of the limited extracellular content of potential thiol substrates. Phospholipid hydroperoxide glutathione peroxidase (PHGPx), originally presumed to be a universal antioxidant enzyme protecting membrane lipids, appears to have adopted a variety of specific roles like silencing lipoxygenases and becoming an enzymatically inactive structural component of the mitochondrial capsule during sperm maturation. Thus, all individual isoenzymes are efficient peroxidases in principle, but beyond their mere antioxidant potential may exert cell- and tissue-specific roles in metabolic regulation, as is evident for PHGPx and may be expected for others.     

Induction of antioxidant enzymes by FAK in a human leukemic cell line, HL-60

We have established several focal adhesion kinase (FAK) cDNA-transfected HL-60 (HL-60/FAK) cells which were highly resistant to oxidative stress-induced apoptosis. To identify target genes that are involved in HL-60/FAK cells, we performed cDNA microarray screening using apoptosis-chip. There, we identified the decrease of glutathione peroxidase (GPx). This result prompted us to investigate the changes of antioxidant enzymes. Here, we demonstrate that lipid peroxidation was suppressed after treatment with hydrogen peroxide in HL-60/FAK cells but not vector-transfected HL-60 (HL-60/Vect) cells. Furthermore, we demonstrate that HL-60/FAK cells have higher basal reactive oxygen species (ROS) levels than the parental HL-60 or HL-60/Vect cells, while ROS accumulation by hydrogen peroxide treatment was almost the same in these cells. Basal activity and mRNA expression of antioxidant enzymes, particularly of GSH reductase (GRe), phospholipid hydroperoxide glutathione peroxidase (PHGPx) were markedly elevated in HL-60/FAK cells. In contrast, GPx and catalase levels were decreased in HL-60/FAK cells. Further, a Src family kinases inhibitor, PP2, suppressed GRe and PHGPx mRNA by inactivation of FAK and c-Src in HL-60/FAK cells. These results suggest that FAK upregulates antioxidant enzymes and suppresses lipid peroxidation, resulting in the anti-apoptotic state for oxidative stress.     

Regulation of enzymatic lipid peroxidation: the interplay of peroxidizing and peroxide reducing enzymes

For a long time lipid peroxidation has only been considered a deleterious process leading to disruption of biomembranes and thus, to cellular dysfunction. However, when restricted to a certain cellular compartment and tightly regulated, lipid peroxidation may have beneficial effects. Early on during evolution of living organisms special lipid peroxidizing enzymes, called lipoxygenases, appeared and they have been conserved during phylogenesis of plants and animals. In fact, a diverse family of lipoxygenase isoforms has evolved starting from a putative ancient precursor. As with other enzymes, lipoxygenases are regulated on various levels of gene expression and there are endogenous antagonists controlling their cellular activity. Among the currently known mammalian lipoxygenase isoforms only 12/15-lipoxygenases are capable of directly oxygenating ester lipids even when they are bound to membranes and lipoproteins. Thus, these enzymes represent the pro-oxidative part in the cellular metabolism of complex hydroperoxy ester lipids. Its metabolic counterplayer, representing the antioxidative part, appears to be the phospholipid hydroperoxide glutathione peroxidase. This enzyme is unique among glutathione peroxidases because of its capability of reducing ester lipid hydroperoxides. Thus, 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase constitute a pair of antagonizing enzymes in the metabolism of hydroperoxy ester lipids, and a balanced regulation of the two proteins appears to be of major cell physiological importance. This review is aimed at summarizing the recent developments in the enzymology and molecular biology of 12/15-lipoxygenase and phospholipid hydroperoxide glutathione peroxidase, with emphasis on cytokine-dependent regulation and their regulatory interplay.     

Biological significance of phospholipid hydroperoxide glutathione peroxidase (PHGPx,GPx4) in mammalian cells

Reactive oxygen species (ROS) are known mediators of intracellular signal cascades. Excessive production of ROS may lead to oxidative stress, loss of cell function, and cell death by apoptosis or necrosis. Lipid hydroperoxides are one type of ROS whose biological function has not yet been clarified. Phospholipid hydroperoxide glutathione peroxidase (PHGPx, GPx4) is a unique antioxidant enzyme that can directly reduce phospholipid hydroperoxide in mammalian cells. This contrasts with most antioxidant enzymes, which cannot reduce intracellular phospholipid hydroperoxides directly. In this review, we focus on the structure and biological functions of PHGPx in mammalian cells. Recently, molecular techniques have allowed overexpression of PHGPx in mammalian cell lines, from which it has become clear that lipid hydroperoxides also have an important function as activators of lipoxygenase and cyclooxygenase, participate in inflammation, and act as signal molecules for apoptotic cell death and receptor-mediated signal transduction at the cellular level.     

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.     

Regulation of cyclooxygenase and 12-lipoxygenase catalysis by phospholipid hydroperoxide glutathione peroxidase in A431 cells

Regulation of arachidonate metabolism in human epidermoid carcinoma A431 cells by phospholipid hydroperoxide glutathione peroxidase (PHGPx) and cytosolic glutathione peroxidase (GPx1) was studied. In order to study the effect of reduced glutathione (GSH) on the catalysis regulation of these oxygenation enzymes, diethyl maleate was used to deplete the intracellular GSH. In the presence of 13-hydroperoxyoctadecadienoic acid, the enzymatic catalysis of cyclooxygenase and 12-lipoxygenase was significantly increased in the GSH-depleted cells. In terms of the inhibitory effect on 12-lipoxygenase, PHGPx was more sensitive to GSH concentrations than GPx1. Inhibition of PHGPx activity by the treatment of cells with antisense oligonucleotide of PHGPx mRNA increased the enzymatic catalysis of both cyclooxygenase and 12-lipoxygenase. In conclusion, the results indicate that catalysis of cyclooxygenase and 12-lipoxygenase in A431 cells was regulated by redox-reaction, and PHGPx seems to play an important role in the controlling of these reactions.     

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.     

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.     

Phospholipid Hydroperoxide Glutathione Peroxidase is a Seleno-Enzyme Distinct from the Classical Glutathione Peroxidase as Evident from Cdna and Amino Acid Sequencing

The primary structure of phospholipid hydroperoxide glutathione peroxidase (PHGPx) was partially elucidated by sequencing peptides obtained by cyanogen bromide cleavage and tryptic digestion and by isolating and sequencing corresponding cDNA fragments covering about 75% of the total sequence. Based on these data PHGPx can be rated as a selenoprotein homologous, but poorly related to classical glutathione peroxidase (GPx). Peptide loops constituting the active site in GPx are, however, strongly conserved in PHGPx. This suggests that the mechanism of action involving an oxidation/reduction cycle of a selenocysteine residue is essentially identical in PHGPx and GPx.     

Isolation and characterization of a novel PHGPx gene in Raphanus sativus

A full-length cDNA encoding putative phospholipid hydroperoxide glutathione peroxidase (PHGPx) was cloned from Raphanus sativus. The cDNA, designated RsPHGPx, includes an open reading frame which encodes 197 amino acid residues. The alignment of amino acid sequences showed that RsPHGPx had the highest sequence homology to plant PHGPx and contained an N-terminal extension characteristic of a mitochondrial targeting peptide. Northern blot analysis indicated that RsPHGPx was constitutively and ubiquitously expressed during radish development, and its expression was differently regulated by various stress conditions. The expression of RsPHGPx in a yeast PHGPx-deletion mutant significantly rescued the mutant sensitivity to oxidation-sensitive linolenic acid, just as the yeast PHGPx3 gene did. This suggested that RsPHGPx encodes a functional PHGPx protein.