Hirotaka Imai

Role of Glutathione Peroxidase 4 in Conjunctival Epithelial Cells

PURPOSEnThe purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in conjunctival epithelial cells.nnnMETHODSnAn immortalized human conjunctival epithelial cell line was used. Cells were transfected with catalase, GPx1, GPx4, SOD1, SOD2, or control siRNA. Knockdown was confirmed by RT-PCR and immunoblotting. The cytotoxicity induced by knockdown of these antioxidant enzymes was examined by assay of LDH activity. Furthermore, evaluations of lipid peroxidation, cellular levels of reactive oxygen species, cell proliferation, and apoptosis were conducted in cells treated with GPx4 or control siRNA. In oxidative stress study, cells treated with GPx4 or control siRNA were applied with hydrogen peroxide or ferric sulfide, and their cytotoxicity was evaluated by assay of LDH activity.nnnRESULTSnSmall interfering RNA of catalase, GPx1, GPx4, SOD1, and SOD2 siRNA remarkably inhibited the mRNA and protein expression of each gene. Knockdown of GPx4 and SOD1 but not catalase, GPx1, and SOD2 significantly induced cytotoxicity. Glutathione peroxidase 4 knockdown increased lipid oxidation and reactive oxygen species. The proliferation of GPx4 siRNA-treated cells was reduced compared with control siRNA-treated cells. Moreover, cell death in GPx4 siRNA-treated cells was characterized by positive staining for annexin V. In an oxidation stress study, GPx4 siRNA knockdown enhanced the cytotoxicity induced by hydrogen peroxide or ferric sulfide.nnnCONCLUSIONnThese results suggest that GPx4 is essential for maintaining oxidative homeostasis and keeping defense against oxidative stress in conjunctival epithelial cells.

Protective Role of Glutathione Peroxidase 4 in Laser-Induced Choroidal Neovascularization in Mice

Purpose To evaluate the influence of glutathione peroxidase 4 (GPx4) expression in retinal pigment epithelium (RPE)/choroid tissue using a mouse model of laser-induced choroidal neovascularization (CNV). Methods In this study, GPx4+/−, GPx4+/+, and GPx4-overexpressing transgenic mice were created for comparison. The mRNA and protein expression of vascular endothelial growth factor (VEGF)-A in RPE/choroid tissue were evaluated before and after CNV induction by laser. Moreover, we investigated the changes in the VEGF-A mRNA level in RPE/choroid tissue in the CNV model that have not been clearly shown previously. Lipid peroxidation in RPE/choroid tissue was evaluated by immunohistochemistry using antibody against 4-hydroxy-2-nonenal. To investigate the protective role of GPx4, the size of laser-induced CNV was compared on day 7 among the mice expressing different levels of GPx4. Results In the laser-induced CNV mouse model, laser treatment reduced the VEGF-A mRNA level in RPE/choroid tissue, while it increased the VEGF-A protein level. Evaluation of VEGF-A expression in RPE/choroid tissue of the GPx4+/−, GPx4+/+, and GPx4 transgenic mice revealed that GPx4 increased the VEGF-A protein level under physiological conditions (i.e., without laser treatment), while GPx4 suppressed the increase in the VEGF-A protein level under pathological conditions (i.e., after CNV induction by laser). In addition, GPx4 reduced the CNV size in a dose-dependent manner in vivo. Conclusions GPx4 suppresses the increase in the VEGF-A protein level, which occurs during the development of pathological CNV, thus partly explaining the protective effect of GPx4 against CNV.

Redox reactions in mammalian spermatogenesis and the potential targets of reactive oxygen species under oxidative stress.

Reduction-oxidation (Redox) reactions are ubiquitous mechanisms for vital activities in all organisms, and they play pivotal roles in the regulation of spermatogenesis as well. Here we focus on 3 redox-involved processes that have drawn much recent attention: the regulation of signal transduction by reactive oxygen species (ROS) such as hydrogen peroxide, oxidative protein folding in the endoplasmic reticulum (ER), and sulfoxidation of protamines during sperm chromatin condensation. The first 2 of these processes are emerging topics in cell biology and are applicable to most living cells, which includes spermatogenic cells. The roles of ROS in signal transduction have been elucidated in the last 2 decades and have received broad attention, most notably from the viewpoint of the proper control of mitotic signals. Redox processes in the ER are important because this is the organelle where secretory and membrane proteins are synthesized and proceed toward their functional structure, so that malfunction of the ER affects not only the involved cells but also the accepting cells of the secreted proteins in multicellular organisms. Sulfoxidation is the third of these processes, and the sulfoxidation of chromatin is a unique process in sperm maturation. During recent sulfoxidase research, GPX4 has emerged as a promising enzyme that plays essential roles in the production of fertile sperm, but the involvement of other redox proteins is also becoming evident. Because the molecules involved in the redox reactions are prone to oxidation, they can be sensitive to oxidative damage, which makes them potential targets for antioxidant therapy.

Lipid Peroxidation-Dependent Cell Death Regulated by GPx4 and Ferroptosis

Glutathione peroxidase 4 (Phospholipid hydroperoxide glutathione peroxidase, PHGPx) can directly reduce phospholipid hydroperoxide. Depletion of GPx4 induces lipid peroxidation-dependent cell death in embryo, testis, brain, liver, heart, and photoreceptor cells of mice. Administration of vitamin E in tissue specific GPx4 KO mice restored tissue damage in testis, liver, and heart. These results indicate that suppression of phospholipid peroxidation is essential for cell survival in normal tissues in mice. Ferroptosis is an iron-dependent non-apoptotic cell death that can elicited by pharmacological inhibiting the cystine/glutamate antiporter, system Xc- (type I) or directly binding and loss of activity of GPx4 (Type II) in cancer cells with high level RAS-RAF-MEK pathway activity or p53 expression, but not in normal cells. Ferroptosis by Erastin (Type I) and RSL3 (RAS-selective lethal 3, Type II) treatment was suppressed by an iron chelator, vitamin E and Ferrostatin-1, antioxidant compound. GPx4 can regulate ferroptosis by suppression of phospholipid peroxidation in erastin and RSL3-induced ferroptosis. Recent works have identified several regulatory factors of erastin and RSL3-induced ferroptosis. In our established GPx4-deficient MEF cells, depletion of GPx4 induce iron and 15LOX-independent lipid peroxidation at 26xa0h and caspase-independent cell death at 72xa0h, whereas erastin and RSL3 treatment resulted in iron-dependent ferroptosis by 12xa0h. These results indicated the possibility that the mechanism of GPx4-depleted cell death might be different from that of ferroptosis induced by erastin and RSL3.

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.

Role of Glutathione Peroxidase 4 in Glutamate-Induced Oxytosis in the Retina

Purpose The purpose of the present study was to investigate the role of glutathione peroxidase 4 (GPx4) in glutamate-induced oxytosis in the retina. Methods For in vitro studies, an immortalized rat retinal precursor cell line R28 was used. Cells were transfected with siRNA specifically silencing GPx4 or with scrambled control siRNA. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal (4-HNE) immunostaining. Cytotoxicity and cell death were evaluated using an LDH activity assay and annexin V staining, respectively. Cells transfected with GPx4 siRNA or control siRNA were treated with glutamate (1 or 2 mM), and the cytotoxicity was evaluated using the LDH activity assay. For in vivo studies, retinal ganglion cell damage was induced by intravitreal injection of 25-mM N-methyl-D-aspartate (NMDA, 2 μL/eye) in GPx4+/+ and GPx4+/− mice. The evaluation of lipid peroxidation (4-HNE immunostaining), apoptosis (TUNEL staining), and cell density in the ganglion cell layer (GCL) were performed at 12 h, 1 day, and 7 days after the NMDA injection. Results GPx4 knockdown significantly increased LDH activity by 13.9-fold (P < 0.01) and increased peroxidized lipid levels by 3.2-fold in R28 cells (P < 0.01). In cells transfected with scrambled control siRNA, treatment with glutamate at 1 or 2 mM did not increase LDH activity; whereas, in cells transfected with GPx4 siRNA, glutamate treatment significantly increased LDH activity (1.52-fold, P < 0.01). GPx4+/− mice exhibited higher levels of lipid peroxidation in retinas treated with NMDA than GPx4+/+ mice (1.26-fold, P < 0.05). GPx4+/− mice had more TUNEL-positive cells induced by NMDA in GCL (1.45-fold, P < 0.05). In addition, the cell density in GCL of GPx4+/− mice was 19% lower than that in GPx4+/+ mice after treatment with NMDA (P < 0.05). Conclusion These results suggest that defective GPx4 expression is associated with enhanced cytotoxicity by glutamate-induced oxytosis in the retina.

Hydrogen peroxide produced by superoxide dismutase SOD-2 activates sperm in Caenorhabditis elegans

Superoxide dismutase (SOD) is a ubiquitous antioxidant enzyme that catalytically converts the superoxide radical to hydrogen peroxide (H2O2). In mammals, high SOD activity is detectable in sperm and seminal plasma, and loss of SOD activity has been correlated with male infertility; however, the underlying mechanisms of sperm infertility remain to be clarified. Here we report that the deletion of two major SOD genes in Caenorhabditis elegans, sod-1 and sod-2, causes sperm activation defects, leading to a significant reduction in brood size. By examining the reactivity to the sperm activation signals Pronase and triethanolamine, we found that sod-1;sod-2 double mutant sperm cells display defects in pseudopod extension. Neither the content nor oxidative modification of major sperm protein, an essential cytoskeletal component for crawling movement, were significantly affected in sod-1;sod-2 mutant sperm. Surprisingly, H2O2, the dismutation product of SOD, could activate sod-1;sod-2 mutant sperm treated with Pronase. Moreover, the H2O2 scavenger ebselen completely inhibited pseudopod extension in wild-type sperm treated with Pronase, and H2O2 could directly induce pseudopod extension in wild-type sperm. Analysis of Pronase-triggered sperm activation in sod-1 and sod-2 single mutants revealed that sod-2 is required for pseudopod extension. These results suggest that SOD-2 plays an important role in the sperm activation of C. elegans by producing H2O2 as an activator of pseudopod extension.

Role of Glutathione Peroxidase 4 in Corneal Endothelial Cells

ABSTRACT Purpose: To investigate the role of glutathione peroxidase 4 (GPx4) in corneal endothelial cells. Materials and Methods: An immortalized human corneal endothelial cell line was used. Cells were transfected with either siRNA specifically silencing GPx4 or scrambled control siRNA. Knockdown was confirmed by Real-time RT-PCR and immunoblotting. Lipid peroxidation was evaluated by 4-hydroxy-2-nonenal immunostaining. Cytotoxicity, cell death, and cell proliferation were evaluated using a lactate dehydrogenase (LDH) activity assay, Annexin V staining, and WST-8, respectively. Furthermore, cells transfected with GPx4 siRNA or control siRNA were treated with hydrogen peroxide or ferrous sulfate, and cytotoxicity was evaluated using the LDH activity assay. Results: The treatment of siRNA decreased the expression of GPx4 at both mRNA and protein levels. The knockdown of GPx4 significantly increased the levels of lipid oxidation and LDH activity. Annexin V-positive cells increased in GPx4 siRNA-treated cells. The proliferation of GPx4 siRNA-treated cells was downregulated compared with that of control siRNA-treated cells. GPx4 knockdown enhanced hydrogen peroxide- and ferrous sulfate-induced cytotoxicity. Conclusion: These results suggest that GPx4 is an important antioxidant enzyme for maintaining redox status and protecting corneal endothelial cells from oxidative stress.

Role of GPx4 in human vascular endothelial cells, and the compensatory activity of brown rice on GPx4 ablation condition

Oxidative stress is implicated in the pathologies of vascular endothelial cells. However, the importance of specific antioxidant enzymes in vascular endothelial cells is not fully understood. The purpose of this study was to elucidate the importance of Glutathione peroxidase 4 (GPx4), and the involvement of ferroptosis on cell death induced by GPx4 loss in human vascular endothelial cells. In addition, we examined the compensatory activity of brown rice on GPx4 ablation condition. Human umbilical vein endothelial cells were transfected with GPx4 or scramble control siRNA. GPx4 knockdown caused the increase in the levels of lipid oxidation, and induced cytotoxicity. On the other hand, α-tocopherol (vitamin E) and extract of brown rice, ameliorated lipid peroxidation, cytotoxicity, and delay of proliferation induced by GPx4 knockdown. Furthermore, ferrostatin-1, inhibitor of ferroptosis, also prevented cytotoxicity and delay of proliferation. In conclusion, our data demonstrated that GPx4 is an essential antioxidant enzyme for protecting lipid peroxidation, and is a regulator of ferroptosis in vascular endothelial cells. Furthermore, vitamin E rich food, such as brown rice, can compensate for GPx4 loss by protecting cells against lipid peroxidation.

Glutathione peroxidase 4 plays an important role in oxidative homeostasis and wound repair in corneal epithelial cells

Oxidative stress is involved in the pathologies of corneal epithelial cells. However, the importance of specific antioxidant enzymes in corneal epithelial cells is not fully understood. The purpose of this study is to elucidate the role of glutathione peroxidase 4 (GPx4) in corneal epithelial cells. For in vitro experiments, an immortalized human corneal epithelial cell line was used. Cytotoxicity measured through LDH activity, lipid peroxidation immunostained for 4‐hydroxynonenal, cell viability, and cell death were compared between cells transfected with either GPx4 siRNA or scrambled control siRNA. In addition, the rescue effects of α‐tocopherol and ferrostatin‐1, a ferroptosis inhibitor, were examined in the cells with deficient GPx4 expression. For in vivo experiments, we applied n‐heptanol on the cornea of GPx4+/+ and GPx4+/− mice to create corneal epithelial wound. The epithelial defect area size was measured up to 48 h after epithelial wound creation. Knockdown of GPx4 strongly induced cytotoxicity and cell death in human corneal epithelial cells. Cell death induced by GPx4 knockdown was characterized by positive staining for both annexin V and propidium iodide, nuclear translocation of AIF, and without activation of caspase 3, and was rescued by α‐tocopherol and ferrostatin‐1. The delayed wound healing of GPx4 siRNA‐transfected cells were ameliorated by α‐tocopherol in vitro. In addition, loss of one GPx4 allele was sufficient to significantly delay the healing of experimental corneal epithelial wounds in vivo. Our results suggest that the antioxidant enzyme GPx4 plays an important role in oxidative homeostasis, cell survival, and wound healing in corneal epithelial cells.