Deletion of the four phospholipid hydroperoxide glutathione peroxidase genes accelerates aging in Caenorhabditis elegans.

The glutathione peroxidase (GPx) family is a major antioxidant enzyme family that catalyzes the reduction of a variety of hydroperoxides. GPxs are divided into selenium- and nonselenium-containing GPxs. Because of their efficient antioxidant activity, which depends on the presence of the amino acid residue selenocysteine, selenium-containing GPxs have been the subject of many studies. However, the physiological roles of the nonselenium GPxs remain unclear. Here, we report that the deletion of phospholipid hydroperoxide glutathione peroxidase (PHGPx) homologues causes accelerated aging that leads to a shortened lifespan in Caenorhabditis elegans. PHGPx is an antioxidant enzyme that directly reduces the phospholipid hydroperoxides generated in biomembranes. The quadruple phgpx mutant gpx-1; gpx-2; gpx-6; gpx-7 developed normally, reached adulthood and reproduced as well as the wild type. However, a lifespan analysis showed that the quadruple phgpx mutant had a short maximum lifespan, with an age-related increase in its mortality rate. The intestine is the primary tissue expressing gpx-1, gpx-2, gpx-6 and gpx-7 in C. elegans, and the expression of gpx-6 is greatly enhanced under starvation conditions. These results suggest that the C. elegans PHGPx homologues have important functions in the regulation of aging, probably by reducing oxidative damage in the intestine.

A congenital muscular dystrophy with mitochondrial structural abnormalities caused by defective de novo phosphatidylcholine biosynthesis.

Congenital muscular dystrophy is a heterogeneous group of inherited muscle diseases characterized clinically by muscle weakness and hypotonia in early infancy. A number of genes harboring causative mutations have been identified, but several cases of congenital muscular dystrophy remain molecularly unresolved. We examined 15 individuals with a congenital muscular dystrophy characterized by early-onset muscle wasting, mental retardation, and peculiar enlarged mitochondria that are prevalent toward the periphery of the fibers but are sparse in the center on muscle biopsy, and we have identified homozygous or compound heterozygous mutations in the gene encoding choline kinase beta (CHKB). This is the first enzymatic step in a biosynthetic pathway for phosphatidylcholine, the most abundant phospholipid in eukaryotes. In muscle of three affected individuals with nonsense mutations, choline kinase activities were undetectable, and phosphatidylcholine levels were decreased. We identified the human disease caused by disruption of a phospholipid de novo biosynthetic pathway, demonstrating the pivotal role of phosphatidylcholine in muscle and brain.

Deficiency of cardiolipin synthase causes abnormal mitochondrial function and morphology in germ cells of Caenorhabditis elegans.

Cardiolipin (CL) is a major membrane phospholipid specifically localized in mitochondria. At the cellular level, CL has been shown to have a role in mitochondrial energy production, mitochondrial membrane dynamics, and the triggering of apoptosis. However, the in vivo role of CL in multicellular organisms is largely unknown. In this study, by analyzing deletion mutants of a CL synthase gene (crls-1) in Caenorhabditis elegans, we demonstrated that CL depletion selectively caused abnormal mitochondrial function and morphology in germ cells but not in somatic cell types such as muscle cells. crls-1 mutants reached adulthood but were sterile with reduced germ cell proliferation and impaired oogenesis. In the gonad of crls-1 mutants, mitochondrial membrane potential was significantly decreased, and the structure of the mitochondrial cristae was disrupted. Contrary to the abnormalities in the gonad, somatic tissues in crls-1 mutants appeared normal with respect to cell proliferation, mitochondrial function, and mitochondrial morphology. Increased susceptibility to CL depletion in germ cells was also observed in mutants of phosphatidylglycerophosphate synthase, an enzyme responsible for producing phosphatidylglycerol, a precursor phospholipid of CL. We propose that the contribution of CL to mitochondrial function and morphology is different among the cell types in C. elegans.

Glutathione peroxidase 4 is required for maturation of photoreceptor cells.

Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells.

Muscle choline kinase beta defect causes mitochondrial dysfunction and increased mitophagy.

Choline kinase is the first step enzyme for phosphatidylcholine (PC) de novo biosynthesis. Loss of choline kinase activity in muscle causes rostrocaudal muscular dystrophy (rmd) in mouse and congenital muscular dystrophy in human, characterized by distinct mitochondrial morphological abnormalities. We performed biochemical and pathological analyses on skeletal muscle mitochondria from rmd mice. No mitochondria were found in the center of muscle fibers, while those located at the periphery of the fibers were significantly enlarged. Muscle mitochondria in rmd mice exhibited significantly decreased PC levels, impaired respiratory chain enzyme activities, decreased mitochondrial ATP synthesis, decreased coenzyme Q and increased superoxide production. Electron microscopy showed the selective autophagic elimination of mitochondria in rmd muscle. Molecular markers of mitophagy, including Parkin, PINK1, LC3, polyubiquitin and p62, were localized to mitochondria of rmd muscle. Quantitative analysis shows that the number of mitochondria in muscle fibers and mitochondrial DNA copy number were decreased. We demonstrated that the genetic defect in choline kinase in muscle results in mitochondrial dysfunction and subsequent mitochondrial loss through enhanced activation of mitophagy. These findings provide a first evidence for a pathomechanistic link between de novo PC biosynthesis and mitochondrial abnormality.

Acidic sphingomyelinase induced by electrophiles promotes proinflammatory cytokine production in human bladder carcinoma ECV-304 cells.

Electrophiles in environmental pollutants or cigarette smoke are high risk factors for various diseases caused by cell injuries such as apoptosis and inflammation. Here we show that electrophilic compounds such as diethyl malate (DEM), methyl mercury and cigarette smoke extracts significantly enhanced the expression of acidic sphingomyelinase (ASMase). ASMase activity and the amount of ceramide of DEM-treated cells were approximately 6 times and 4 times higher than these of non-treated cells, respectively. Moreover, we found that DEM pretreatment enhanced the production of IL-6 induced by TNF-α. Knockdown of ASMase attenuated the enhancement of TNF-α-dependent IL-6 production. On the other hand, enhancement of TNF-α-induced IL-6 production was observed in ASMase-overexpressing cells without DEM. Fractionation of the lipid raft revealed that the TNF receptor 1 (TNFR1) was migrated into the lipid raft in DEM-treated cells or ASMase-overexpressing cells. The TNF-α-induced IL-6 expression required the clustering of TNFR1 since IL-6 expression were decreased by the destruction of the lipid raft with filipin. These results demonstrated a new role for ASMase in the acceleration of the production of TNF-induced IL-6 as a pro-inflammatory cytokine and indicated that electrophiles could potentiate inflammation response by up-regulating of ASMase expression following formation of lipid rafts.

Depletion of selenoprotein GPx4 in spermatocytes causes male infertility in mice.

Phospholipid hydroperoxide glutathione peroxidase (GPx4) is an intracellular antioxidant enzyme that directly reduces peroxidized phospholipids. GPx4 is strongly expressed in the mitochondria of testis and spermatozoa. We previously found a significant decrease in the expression of GPx4 in spermatozoa from 30% of infertile human males diagnosed with oligoasthenozoospermia (Imai, H., Suzuki, K., Ishizaka, K., Ichinose, S., Oshima, H., Okayasu, I., Emoto, K., Umeda, M., and Nakagawa, Y. (2001) Biol. Reprod. 64, 674-683). To clarify whether defective GPx4 in spermatocytes causes male infertility, we established spermatocyte-specific GPx4 knock-out mice using a Cre-loxP system. All the spermatocyte-specific GPx4 knock-out male mice were found to be infertile despite normal plug formation after mating and displayed a significant decrease in the number of spermatozoa. Isolated epididymal GPx4-null spermatozoa could not fertilize oocytes in vitro. These spermatozoa showed significant reductions of forward motility and the mitochondrial membrane potential. These impairments were accompanied by the structural abnormality, such as a hairpin-like flagella bend at the midpiece and swelling of mitochondria in the spermatozoa. These results demonstrate that the depletion of GPx4 in spermatocytes causes severe abnormalities in spermatozoa. This may be one of the causes of male infertility in mice and humans.

Identification of a responsible promoter region and a key transcription factor, CCAAT/enhancer-binding protein epsilon, for up-regulation of PHGPx in HL60 cells stimulated with TNF alpha.

In the present study we investigated promoter regions of the PHGPx [phospholipid hydroperoxide GPx (glutathione peroxidase)] gene and transcription factors involved in TNFalpha (tumour necrosis factor alpha)-induced up-regulation of PHGPx in non-differentiated HL60 cells. Non-differentiated HL60 cells displayed up-regulation of non-mitochondrial and mitochondrial PHGPx mRNA in response to TNFalpha stimulation. The promoter activity was up-regulated by TNFalpha stimulation in cells transfected with a luciferase reporter vector encoding the region from -282 to -123 of the human PHGPx gene compared with the non-stimulated control. The up-regulated promoter activity was effectively abrogated by a mutation in the C/EBP (CCAAT/enhancer-binding protein)-binding sequence in this region. ChIP (chromatin immunoprecipitation) assays demonstrated that C/EBPepsilon bound to the -247 to -34 region in HL60 cells, but C/EBPalpha, beta, gamma and delta did not. The binding of C/EBPepsilon to the promoter region was increased in HL60 cells stimulated with TNFalpha compared with that of the non-stimulated control. An increased binding of nuclear protein to the C/EBP-binding sequence was observed by EMSA (electrophoretic mobility-shift assay) in cells stimulated with TNFalpha, and it was inhibited by pre-treatment with an anti-C/EBPepsilon antibody, but not with other antibodies. The C/EBPepsilon mRNA was expressed in PMNs (polymorphonuclear cells), non-differentiated HL60 cells and neutrophil-like differentiated HL60 cells displaying TNFalpha-induced up-regulation of PHGPx mRNA, but not in macrophage-like differentiated HL60 cells, HEK-293 cells (human embryonic kidney-293 cells) and other cell lines exhibiting no up-regulation. The up-regulation of PHGPx mRNA, however, was detected in HEK-293 cells overexpressing C/EBPepsilon as a result of TNFalpha stimulation. These results indicate that C/EBPepsilon is a critical transcription factor in TNFalpha-induced up-regulation of PHGPx expression.

Identification of the positive regulatory and distinct core regions of promoters, and transcriptional regulation in three types of mouse phospholipid hydroperoxide glutathione peroxidase.

Phospholipid hydroperoxide glutathione peroxidase (PHGPx) is transcribed into three types of mRNA, mitochondrial, non-mitochondrial and nucleolar types, from one gene by alternative transcription using different first exons, Ia and Ib. We investigated the regulatory mechanisms of the expressions of the three types of PHGPx using promoter analysis with luciferase as the reporter gene and electrophoretical mobility shift analysis. Here we report a draft of the positive regulatory region and the core promoter regions of PHGPx in several cell lines. From promoter deletion analysis we identified the three distinct core regions of mitochondrial PHGPx, non-mitochondrial PHGPx and nucleolar PHGPx. The core promoter activity of non-mitochondrial PHGPx was high in L929 cells, but relatively low for mitochondrial and nucleolar PHGPx. We also identified the positive regulatory region of mitochondrial PHGPx by deletion and mutation analysis of 5'-flanking regions of mitochondrial PHGPx. This region could regulate the promoter activity of non-mitochondrial PHGPx; however, up-regulation by this region was normally suppressed by the upstream region in somatic cells. Electrophoretical mobility shift analysis demonstrated that a specific transcription factor complex bound to this region in adult testis, but not in young testis and different sizes of complexes bound to this region between testis and brain.

Up-regulation of phospholipid hydroperoxide glutathione peroxidase in rat casein-induced polymorphonuclear neutrophils.

Antioxidant enzymes play key roles in the protection of cells from oxidative damage. Little is known, however, about the expression of antioxidants and/or their roles in PMNs (polymorphonuclear leucocytes), which are thought to suffer from oxidative stress in an inflammation site. In the present paper, we report on the regulation of expression of PHGPx (phospholipid hydroperoxide glutathione peroxidase) and cGPx (cytosolic glutathione peroxidase) in rat PMNs in the inflammation site. PHGPx mRNA levels were much lower in casein-induced peritoneal and carrageenan-induced pleural PMNs just after their collection than in peripheral PMNs. cGPx mRNA was also reduced in the casein-induced PMNs, but not in carrageenan-induced PMNs. Both enzymes with decreased levels in the casein-induced PMNs were up-regulated during further 24 h cultivation in vitro and in vivo, with elevation of their protein levels and activities, and reduction of intracellular peroxides. Up-regulation of PHGPx mRNA was attenuated by cycloheximide, a protein synthesis inhibitor, and this effect was cancelled by culturing the cells in the conditioned medium of the cultured casein-induced PMNs. This latter effect was attenuated by pre-treatment with anti-GRO (growth-regulated oncogene) antibody. Recombinant rat GRO could also induce the up-regulation in the presence of cycloheximide, demonstrating that GRO may play an important role in the PHGPx up-regulation of casein-induced PMNs. Production of the lipid mediators leukotriene B4 and 5-HETE (5-hydroxyeicosatetraenoic acid) was decreased in the cultured casein-induced PMNs exhibiting PHGPx up-regulation. The evidence obtained indicates that PHGPx activity in the activated PMNs would be related to the appearance of the intrinsic function of PMNs in the inflammatory site.

An abortive apoptotic pathway induced by singlet oxygen is due to the suppression of caspase activation.

Singlet oxygen causes the cytotoxic process of tumour cells in photodynamic therapy. The mechanism by which singlet oxygen damages cells is, however, not fully understood. To address this issue, we synthesized and used two types of endoperoxides, MNPE (1-methylnaphthalene-4-propionate endoperoxide) and NDPE (naphthalene-1,4-dipropionate endoperoxide), that generate defined amounts of singlet oxygen at 37 degrees C with similar half lives. MNPE, which is more hydrophobic than NDPE, induced the release of cytochrome c from mitochondria into the cytosol and exhibited cytotoxicity, but NDPE did not. RBL cells, a rat basophil leukaemia-derived line, that overexpress phospholipid hydroperoxide glutathione peroxidase in mitochondria were found to be highly resistant to the cytotoxic effect of MNPE. MNPE treatment induced much less DNA ladder formation and nuclear fragmentation in cells than etoposide treatment, even though these treatments induced a similar extent of cellular damage. Singlet oxygen inhibited caspase 9 and 3 activities directly and also suppressed the activation of the caspase cascade. Collectively, these data suggest that singlet oxygen triggers an apoptotic pathway by releasing cytochrome c from mitochondria via the peroxidation of mitochondrial components and results in cell death that is different from typical apoptosis, because of the abortive apoptotic pathway caused by impaired caspase activation.

Role of calcium-induced mitochondrial hydroperoxide in induction of apoptosis of RBL2H3 cells with eicosapentaenoic acid treatment.

Eicosapentaenoic acid (EPA) was previously shown to induce caspase-independent apoptosis in rat basophilic leukemia cells (RBL2H3 cells) by translocation of apoptosis-inducing factor (AIF) [Free Radic Res (2005) 39, 225-235]. Here, we attempted to investigate the mechanism of EPA-induced apoptosis. A rapid and sustained increase in calcium was observed in mitochondria at 2 h after the addition of EPA prior to apoptosis. Coincidently, hydroperoxide was generated in the mitochondria after exposure to EPA. Production of mitochondrial hydroperoxide was significantly reduced by ruthenium red, an inhibitor of mitochondrial calcium uniporter, and BAPTA-AM, a cytoplasmic calcium chelator, indicating that generation of hydroperoxide is triggered by an accumulation of calcium in the mitochondria. The production of mitochondrial hydroperoxide was markedly attenuated by overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in the mitochondria. Apoptosis was therefore, significantly prevented through inhibition of mitochondrial hydroperoxide generation with mitochondrial PHGPx, ruthenium red or BAPTA-AM. However, accumulation of calcium in the mitochondria was not prevented by mitochondrial PHGPx although apoptosis was blocked, indicating that elevated calcium does not directly induce apoptosis. Taken together, our results show that calcium-dependent hydroperoxide accumulation in the mitochondria is critical in EPA-induced apoptosis.

Involvement of hydroperoxide in mitochondria in the induction of apoptosis by the eicosapentaenoic acid.

Eicosapentaenoic acid (EPA) induced apoptosis of rat basophilic leukemia cells (RBL2H3 cells), whereas 100 microM linoleic acid (LA) had no significant effect. Cytochrome c was released at 4 h. Apoptosis was detected at 6 h after exposure to EPA and docosahexaenoic acid (DHA), and preceded the activation of caspase-3. Liberation of apoptosis-inducing factor (AIF) from mitochondria and its translocation into the nucleus were observed at 4 h. A broad-specificity caspase inhibitor, z-VAD-fmk, failed to suppress the apoptosis, suggesting that EPA induced caspase-independent apoptosis. On other hand, a poly (ADP-ribose) polymerase-1 (PARP-1) inhibitor that blocks AIF translocation to the nucleus suppressed EPA-induced apoptosis. The level of hydroperoxide in the cells and mitochondria increased at the early phase of apoptosis within 2 h. On the contrary, elevation of hydroperoxide in mitochondria was not observed after treatment with LA. The EPA-induced apoptosis was abolished by prevention of the hydroperoxide elevation in mitochondria via overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (PHGPx). Neither cytochrome c nor AIF were released from mitochondria in the mitochondrial PHGPx-overexpressing cells. EPA also induced apoptosis in HeLa cells, but not in L929 or RAW264.7 cells. Enhancement of the hydroperoxide level in mitochondria was found in the EPA-sensitive HeLa cells after treatment with EPA, whereas no such enhancement was observed in the apoptosis-resistant L929 and RAW264.7 cells. These results suggest that the generation of hydroperoxide in mitochondria induced by EPA is associated with AIF release from mitochondria and the induction of apoptosis.

Nrf2 up-regulates the induction of acidic sphingomyelinase by electrophiles.

Acidic sphingomyelinase (ASMase) catalyses the generation of ceramide from sphingomyelin. Ceramide is a lipid mediator and is implicated in mediating and regulating various cellular processes including cell proliferation, differentiation, stress response and inflammation. We have previously reported that electrophiles including diethyl maleate (DEM), heavy metals and cigarette smoke extracts induced ASMase expression in human bladder carcinoma ECV-304 cells, but the mechanism of ASMase mRNA induction by electrophiles remains unknown. In this study, we clarified the involvement of NF-E2-related factor 2 (Nrf2) in the induction of ASMase mRNA by DEM. Promoter analysis using a series of deletion mutants of the human ASMase gene showed that ARE-like element1 located in a region between -200 and -160 bp upstream of the transcription start point is mainly a DEM-responsive element. Moreover, an electrophoretic mobility shift assay using ARE-like element1 revealed that Nrf2 is a candidate transcription factor that binds to ARE-like element1 in response to DEM. Finally, alteration of Nrf2 expression by overexpression and knockdown could regulate the induction of ASMase mRNA by DEM. This is the first evidence that supports the possibility that sphingolipid metabolism is affected via the induction of ASMase by the Nrf2 pathway.

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.

A subset of newly synthesized polypeptides in mitochondria from human endothelial cells exposed to hydroperoxide stress.

The synthesis of 40 polypeptides in mitochondria was found to be stimulated after transient exposure of human endothelial cells to sublethal levels of hydroperoxides, such as H(2)O(2), using comparative two-dimensional polyacrylamide gel electrophoresis. Eleven proteins were identified; these include 60 kDa heat shock protein (HSP60), a mitochondrial type of 70 kDa HSP (mtHSP70), manganese-dependent superoxide dismutase (MnSOD), three metabolic enzymes in citric acid cycle, two components for respiratory chain complexes, a ribosomal protein for translation in mitochondria (RM12), and an unnamed protein. These proteins are involved in reduction-oxidation and protein biogenesis, suggesting that their synthesis, which is triggered under oxidative stress conditions, is aimed at playing a defensive role in mitochondria. Moreover, mtHSP70, HSP60, MnSOD, and RM12 were revealed as their respective precursor proteins with mitochondrial targeting sequences. The preproteins of HSP60 and mtHSP70 were transiently accumulated in mitochondria after the removal of H(2)O(2) in a processing competent state, while the accumulated preprotein of MnSOD localized inside mitochondria and remained unchanged. Membrane potential of mitochondria and cellular ATP levels were unchanged under these conditions. Taken together, these results suggest that hydroperoxide stress leads to preprotein accumulation, possibly due to the impairment of the protein-processing system in mitochondria, independent of membrane potential dissipation and ATP depletion.

Initiation of apoptotic signal by the peroxidation of cardiolipin of mitochondria.

Overexpression of phospholipid hydroperoxide glutathione peroxidase (PHGPx) in mitochondria of RBL2H3 cells (M15 cells) prevented the release of cytochrome c (cyt.c), the activation of caspase-3, and apoptosis caused by 2-deoxyglucose (2DG), whereas cells overexpressing nonmitochondrial PHGPx(L9) and control (S1) cells were induced to apoptosis. Hydro-peroxide levels in mitochondria of L9 and S1 cells were significantly enhanced by 2DG-induced apoptosis. In contrast, generation of hydroperoxide in mitochondria was protected in M15 cells, which also showed resistance to apoptosis by etoposide, staurosporine, UV irradiation, cycloheximide, and actinomycin D, stimuli that induce apoptosis by the liberation of cyt.c from mitochondria. Cyt.c preferentially binds to the monolayer of cardiolipin (CL), the specific phospholipid of the inner membrane of mitochondria. The amount of cyt.c bound to the monolayer of cardiolipin hydroperoxide (CL-OOH) was much lower than that bound to CL. Cyt.c bound to liposome containing CL was released by peroxidation with a radical initiator. Adenine nucleotide translocator (ANT), which regulates the opening and closing the permeability transition (PT) pore, potentially was inactivated in apoptosis-induced S1 cells 4 h after the addition of 2DG, coincidentally with cyt.c release from mitochondria. ANT activity was suppressed by the fusion of isolated mitochondria with liposomes containing CL-OOH. ANT activity was expressed in proteoliposomes containing 10% CL, but it was competitively inhibited by the addition of CL-OOH. This study suggests that CL peroxidation might have an initiating role in the liberation of cyt.c from the inner membrane, and in the opening of the PT pore via inactivation of ANT. Mitochondrial PHGPx might play a role as an anti-apoptotic factor by protecting CL and reducing CL-OOH.

Alteration of gene expressions by the overexpression of mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx).

To determine the effect on gene expression of trace levels of reactive oxygen species from mitochondria, we used the mRNA differential display technique to compare gene expression in two cell lines: M15, which overexpresses mitochondrial phospholipid hydroperoxide glutathione peroxidase (mtPHGPx), in rat basophilic leukemia RBL-2H3 cells, and a control cell line, S1. We isolated 27 differentially expressed genes, including 10 previously unreported sequences. These genes included cytoskeletal proteins (beta-tubulin, nonmuscle myosin alkali light chain, and vimentin), growth or proliferation regulators [growth differentiation factor 1 (Gdf-1), Rap1a, and inhibitor of growth 3 (Ing3)], and others. Although the expression of most of the isolated genes did not respond to ROS (hydrogen peroxide) or antioxidant (pyrolidine dithiocarbamate) treatment, the expression of Gdf-1 was downregulated by hydrogen peroxide treatment. Thus, low levels of ROS produced in mitochondria during normal cellular metabolism can modulate gene expression.

[Metabolism and biological function of cardiolipin].

Cardiolipin (CL) is a phospholipid, which is exclusively located in mitochondria, and has a unique structure that consists of 2 phosphate residues and 4 kinds of fatty acyl chains. Cardiolipin plays an important role in regulating various kinds of mitochondrial proteins such as electron transport complexes, carrier proteins and phosphate kinases, and is also essential for the organization of particular mitochondrial structures such as cristae and contact sites. Mitochondrial phospholipase D hydrolyzes CL to produce phosphatidic acid, which is required for mitochondrial fusion. Oxidative stress-induced peroxidation of CL occurs because CL is rich in polyunsaturated fatty acids, especially linoleic acid. Accumulation of CL hydroperoxide (CLOOH) triggers the initiation of apoptosis. Formation of CLOOH causes the release of proapoptotic factors such as cytochrome c from the inner mitochondrial membrane and triggers opening of the permeability transition pore. Levels of CL decrease in the heart following ischemia or disease. Apoptosis is enhanced in temperature-dependent mutant cells whose amounts of CL reduce to half when compared to that of wild type cells. Low levels of CL cause the accumulation of CLOOH and enhance sensitivity to apoptosis. Accumulation of CLOOH in mitochondria causes instability of the membrane, because swelling of mitochondria is induced by the presence of CLOOH in the membrane and is significantly enhanced in CLOOH-loaded mitochondria by the addition of inducer of swelling.