Glutathione Peroxidase-1 Knockout Facilitates Memory Impairment Induced by ß-Amyloid (1-42) in Mice via Inhibition of PKC ßII-Mediated ERK Signaling; Application with Glutathione Peroxidase-1 Gene-Encoded Adenovirus Vector.

A growing body evidence suggests that selenium (Se) deficiency is associated with an increased risk of developing Alzheimer's disease (AD). Se-dependent glutathione peroxidase-1 (GPx-1) of a major antioxidant enzyme, and the most abundant isoform of GPx in the brain. In the present study, we investigated whether GPx-1 is protective against memory impairments induced by beta-amyloid (Aß) (1-42) in mice. As the alteration of protein kinase C (PKC)-mediated ERK activation was recognized in the early stage of AD, we examined whether the GPx-1 gene modulates Aß (1-42)-induced changes in PKC and ERK levels. We observed that Aß (1-42) treatment (400 pmol, i.c.v.) significantly decreased PKC ßII expression in the hippocampus of mice. Aß (1-42)-induced neurotoxic changes [i.e., oxidative stress (i.e., reactive oxygen species, 4-hydroxy-2-noneal, and protein carbonyl), reduced PKC ßII and phospho-ERK expressions, and memory impairment under Y-maze and passive avoidance test] were more pronounced in GPx-1 knockout than in wild type mice. Importantly, exposure to a GPx-1 gene-encoded adenovirus vector (Adv-GPx-1) significantly increased GPx-1 mRNA and GPx activity in the hippocampus of GPx-1 knockout mice. Adv-GPx-1 exposure also significantly blocked the neurotoxic changes induced by Aß (1-42) in GPx-1 knockout mice. Treatment with ERK inhibitor U0126 did not significantly change Adv-GPx-1-mediated attenuation in PKC ßII expression. In contrast, treatment with PKC inhibitor chelerythrine (CHE) reversed Adv-GPx-1-mediated attenuation in ERK phosphorylation, suggesting that PKC ßII-mediated ERK signaling is important for Adv-GPx-1-mediated potentials against Aß (1-42) insult. Our results suggest that treatment with the antioxidant gene GPx-1 rescues Aß (1-42)-induced memory impairment via activating PKC ßII-mediated ERK signaling.

Loss of glutathione peroxidase 3 induces ROS and contributes to prostatic hyperplasia in Nkx3.1 knockout mice.

BACKGROUND: Glutathione peroxidase 3 (Gpx3) protects cells from oxidative stress, and its reduced expression in human prostate cancer has been reported. OBJECTIVES: We hypothesized that Gpx3 might play an important role in the development of prostatic intraepithelial neoplasia (PIN), a pre-cancerous state of the prostate, and aimed to highlight the underlying molecular mechanism. MATERIALS AND METHODS: The following double-knockout mice Nkx3.1-/-; Gpx3+/+, Nkx3.1-/-; Gpx3+/-, Nkx3.1-/-; Gpx3-/- were produced. Randomly divided animals were weighed, and their genitourinary tract (GUT) weights were determined after euthanasia at 4, 8, and 12 months. The mRNA expression of the genes involved in oxidative stress and Wnt signaling was analyzed in the prostate. Histopathology, ROS, and superoxide dismutase (SOD) activities were also measured. RESULTS: Loss of Gpx3 did not affect body weight and GUT weight in Nkx3.1 knockout mice. The mRNA expression of SOD3, iNOS, Hmox, and CISD2, which are associated with oxidative stress, was increased in Nkx3.1-/-; Gpx3-/- mice at 4 months but decreased at 8 and 12 months. There was no change in ß-catenin and its targets associated with Wnt signaling. Increased ROS and decreased SOD activity were observed in Nkx3.1-/-; Gpx3-/- mice at 12 months of age. The histopathologic score and epithelium thickness were increased, and lumen area was decreased in Gpx3 knockout mice. DISCUSSION AND CONCLUSIONS: Gpx3 loss increased the hyperplasia of PIN in the pre-cancerous stage of the prostate. Loss of Gpx3 induced oxidative stress. Histopathologically, no invasive carcinoma was identified, and Gpx3 loss did not increase Wnt/ß-catenin signaling. Further research on the role of GPX3 in the transition of PIN to invasive carcinoma is needed. We show, for the first time, that the antioxidant enzyme GPX3 plays a vital role in inhibiting hyperplasia in the PIN stage of the prostate gland in vivo.

Hypermethioninemia induces memory deficits and morphological changes in hippocampus of young rats: implications on pathogenesis.

The aim of this study was to investigate the effect of the chronic administration of methionine (Met) and/or its metabolite, methionine sulfoxide (MetO), on the behavior and neurochemical parameters of young rats. Rats were treated with saline (control), Met (0.2-0.4 g/kg), MetO (0.05-0.1 g/kg), and/or a combination of Met + MetO, subcutaneously twice a day from postnatal day 6 (P6) to P28. The results showed that Met, MetO, and Met + MetO impaired short-term and spatial memories (P < 0.05), reduced rearing and grooming (P < 0.05), but did not alter locomotor activity (P > 0.05). Acetylcholinesterase activity was increased in the cerebral cortex, hippocampus, and striatum following Met and/or MetO (P < 0.05) treatment, while Na+, K+-ATPase activity was reduced in the hippocampus (P < 0.05). There was an increase in the level of thiobarbituric acid reactive substances (TBARS) in the cerebral cortex in Met-, MetO-, and Met + MetO-treated rats (P < 0.05). Met and/or MetO treatment reduced superoxide dismutase, catalase, and glutathione peroxidase activity, total thiol content, and nitrite levels, and increased reactive oxygen species and TBARS levels in the hippocampus and striatum (P < 0.05). Hippocampal brain-derived neurotrophic factor was reduced by MetO and Met + MetO compared with the control group. The number of NeuN-positive cells was decreased in the CA3 in Met + MetO group and in the dentate gyrus in the Met, MetO, and Met + MetO groups compared to control group (P < 0.05). Taken together, these findings further increase our understanding of changes in the brain in hypermethioninemia by elucidating behavioral alterations, biological mechanisms, and the vulnerability of brain function to high concentrations of Met and MetO.

Deficiency of glutathione peroxidase-1 and catalase attenuated diet-induced obesity and associated metabolic disorders.

AIMS: Oxidative stress has been considered to contribute to the development of obesity-related metabolic disorders including insulin resistance. To the contrary, deficiency of an anti-oxidizing enzyme, glutathione peroxidase (GPx)-1, was reported to enhance insulin signaling, suggesting that oxidative stress may inhibit the development of type 2 diabetes. However, the beneficial effects of the absence of GPx-1 in metabolic homeostasis, including body weight control, have not yet been clearly manifested. To clarify the relationship between oxidative stress and obesity-related metabolic disorders, we investigated another mouse deficient with both GPx-1 and catalase (Cat). METHODS: C57BL/6J wild-type and GPx-1-/- × Cat-/- mice were fed with a high-fat diet (60% fat) or a normal chow diet for 16 weeks and were investigated for metabolic and histological studies. RESULTS: Body weight gain was significantly reduced, and glucose metabolism as well as hepatic steatosis was obviously improved in the GPx-1-/- × Cat-/- mice. The serum levels of insulin and total cholesterol were also significantly lowered. For the underlying mechanism, inflammation was attenuated and expression of markers for fat browning was enhanced in the visceral white adipose tissues. CONCLUSIONS: Oxidative stress due to deficiency of GPx-1 and Cat may improve obesity-related metabolic disorders through attenuation of inflammation and fat browning.

Glutathione peroxidase-1 gene rescues cocaine-induced conditioned place preference in mice by inhibiting σ-1 receptor expression.

The aim of this study was to investigate whether the glutathione peroxidase-1 gene (GPx-1) affects cocaine-induced conditioned place preference (CPP) using a mouse model. Cocaine-induced CPP was accompanied by an increase in the level of σ-1 receptor in the nucleus accumbens (NAc). This phenomenon was more pronounced in the GPx-1 gene knockout (GPx-1 KO) than in wild type (WT) mice. In contrast, the CPP and expression of σ-1 receptor were much less pronounced in GPx-1-overexpressing transgenic (GPx-1 TG) mice than non-transgenic (non-TG) mice. Treatment of the mice with BD1047, a σ-1 receptor antagonist, significantly attenuated both cocaine-induced CPP and c-Fos-immunoreactivity (c-Fos-IR) in WT and GPx-1 KO mice, although the effects were more evident in the latter group. Despite the protective effects of BD1047 on cocaine-induced CPP and c-Fos in non-TG mice, there were no additional protective effects in cocaine-treated GPx-1 TG mice, indicating that the σ-1 receptor is a critical target for GPx-1-mediated psychoprotective activity. Overall, our results suggest that GPx-1 attenuates cocaine-induced CPP via inhibition of σ-1 receptor expression.

Downregulation of microRNA-196a inhibits stem cell self-renewal ability and stemness in non-small-cell lung cancer through upregulating GPX3 expression.

Studies have reported a high expression profile of microRNA-196a (miR-196a) in many cancers, which potently plays important roles in carcinogenesis. However, the involvement of miR-196a in affecting non-small cell lung cancer (NSCLC) carcinogenesis still remains uncertain. NSCLC-related differentially expressed genes were retrieved for this study according to the microarray-based analysis, which demonstrated that miR-196a may be involved in NSCLC progression via regulation of the Jun N-terminal kinase (JNK) pathway by targeting glutathione peroxidase 3 (GPX3). Hence, this study aimed to explore the relationship among miR-196a, GPX3, and the JNK pathway and to investigate its functional regulations in NSCLC. Initially, highly-expressed miR-196a and lowly-expressed GPX3 were determined in NSCLC tissues and cells. Next, the NSCLC cells were manipulated with a series of mimic, inhibitor or shRNA to investigate the impact of miR-196a and GPX3 on CSC viability, proliferation, self-renewal ability and stemness. The in vivo effect of miR-196a was measured in nude mice xenografted with NSCLC cells. The results demonstrated that downregulation of miR-196a and restoration of GPX3 inhibited CSC viability, proliferation, self-renewal ability, stemness and tumorigenicity. Meanwhile, the underlying relationship among miR-196a, GPX3 and JNK pathway was explored by treatment with the JNK pathway inhibitor (SP600125), or sh-GPX3. Downregulated miR-196a and upregulated GPX3 could elevate the GPX3 protein expression and reduce the extent of JNK and c-Jun phosphorylation. Taken together, miR-196a promotes the development of NSCLC via activation of the JNK pathway through down-regulation of GPX3 and serve as a potential therapeutic target in NSCLC.

Overexpression of glutathione peroxidase-1 attenuates cocaine-induced reproductive dysfunction in male mice by inhibiting nuclear factor κB.

Since reproductive toxicity is associated with oxidative stress, nuclear factor κB (NFκB), a redox-sensitive transcription factor, may be involved in the reproductive dysfunction induced by the abusive drug, such as cocaine. In the present study, we investigated whether NFκB mediates cocaine-induced reproductive dysfunction in male mice, and whether glutathione peroxidase (GPx)-1, a well-known enzymatic antioxidant, modulates NFκB activity to affect this reproductive dysfunction. Cocaine treatment significantly increased nuclear translocation of NFκB and its DNA binding activity in the testis of mice. Treatment with cocaine resulted in a significant increase in sperm abnormality, and in significant decreases in the sperm viability and sperm level. Furthermore, cocaine significantly reduced hypothalamic gonadotropin-releasing-hormone expression and plasma testosterone level. These alterations were more pronounced in the GPx-1 knockout (GPx-1 KO) than wild type (WT) mice, and they were less pronounced in GPx-1 overexpressing transgenic (GPx-1 TG) than in non-transgenic (non-TG) mice. Pyrrolidine dithiocarbamate (PDTC), an NFκB inhibitor, was more effective in attenuating cocaine-induced reproductive toxicity in GPx-1 KO than in WT mice. Although PDTC treatment was also significantly protective against the reproductive toxicity in non-TG mice, PDTC did not show additional positive effects against the protective potential mediated by GPx-1 overexpression in mice. Therefore, our results suggest that GPx-1 gene is a protective factor in response to reproductive dysfunction induced by cocaine in male mice, and that NFκB is a critical mediator of protective activity of GPx-1 gene in our experimental conditions.

Unsolved mysteries: How does lipid peroxidation cause ferroptosis?

Ferroptosis is a cell death process driven by damage to cell membranes and linked to numerous human diseases. Ferroptosis is caused by loss of activity of the key enzyme that is tasked with repairing oxidative damage to cell membranes-glutathione peroxidase 4 (GPX4). GPX4 normally removes the dangerous products of iron-dependent lipid peroxidation, protecting cell membranes from this type of damage; when GPX4 fails, ferroptosis ensues. Ferroptosis is distinct from apoptosis, necroptosis, necrosis, and other modes of cell death. Several key mysteries regarding how cells die during ferroptosis remain unsolved. First, the drivers of lipid peroxidation are not yet clear. Second, the subcellular location of lethal lipid peroxides remains an outstanding question. Finally, how exactly lipid peroxidation leads to cell death is an unsolved mystery. Answers to these questions will provide insights into the mechanisms of ferroptotic cell death and associated human diseases, as well as new therapeutic strategies for such diseases.

Impact of Glutathione Peroxidase-1 (Gpx1) Genotype on Selenoenzyme and Transcript Expression When Repleting Selenium-Deficient Mice.

Glutathione peroxidase (Gpx1) is the major selenoprotein in most tissues in animals. Knockout (KO) of Gpx1 decreases Gpx1 activity to near zero and substantially reduces liver selenium (Se) levels, but has no overt effects in otherwise healthy mice. To investigate the impact of deletion of Gpx1 on Se metabolism, Se flux, and apparent Se requirements, KO, Gpx1 heterozygous (Het), and Gpx1 wild-type (WT) mice were fed Se-deficient diet for 17 weeks, then repleted with graded levels of Se (0-0.3 µg Se/g as Na2SeO3) for 7 days, and selenoprotein activities and transcripts were determined in blood, liver, and kidney. Se deficiency decreased the activities of plasma Gpx3, liver Gpx1, liver Txnrd, and liver Gpx4 to 3, 0.3, 11, and 50% of WT Se-adequate levels, respectively, but the Gpx1 genotype had no effect on growth or changes in activity or expression of selenoproteins other than Gpx1. Se repletion increased selenoprotein transcripts to Se-adequate levels after 7 days; Se response curves and apparent Se requirements for selenoprotein transcripts were similar to those observed in studies starting with Se-adequate mice. With short-term Se repletion, selenoenzyme activities resulted in three Se response curve patterns: (1) liver and kidney Gpx1, Gpx4, and Txnrd activities were sigmoidal or hyperbolic with breakpoints (0.08-0.19 µg Se/g) that were double those observed in studies starting with Se-adequate mice; (2) red blood cell Gpx1 activity was not significantly changed; and (3) plasma Gpx3 activity only increased substantially with 0.3 µg Se/g. Plasma Gpx3 is secreted from kidney. In this short-term study, kidney Gpx3 mRNA reached plateau levels at 0.1 µg Se/g, and other kidney selenoenzyme activities reached plateau levels at ≤ 0.2 µg Se/g, so sufficient Se should have been present in kidney. Thus, the delayed increase in plasma Gpx3 activity suggests that newly synthesized and secreted kidney Gpx3 is preferentially retained in kidney or rapidly cleared by binding to basement membranes in kidney or in other tissues. This repletion study shows that loss of capacity to incorporate Se into Gpx1 in Gpx1 KO mice does not dramatically alter expression of other Se biomarkers, nor the short-term flux of Se from intestine to liver to kidney.

Alterations in neuronal control of body weight and anxiety behavior by glutathione peroxidase 4 deficiency.

Elevated levels of oxidative stress and neuronal inflammation in the hypothalamus or ventral midbrain, respectively, represent common denominators for obesity and Parkinson's Disease (PD). However, little is known about defense mechanisms that protect neurons in these regions from oxidative damage. Here, we aimed to assess whether murine Gpx4, a crucial antioxidant enzyme that protects neurons from membrane damage and ferroptosis, is critical for the protection from neuronal inflammation in two distinct pathophysiologic diseases, namely metabolic dysfunction in diet-induced obesity or PD. Gpx4 was deleted from either AgRP or POMC neurons in the hypothalamus, essential for metabolic homeostasis, or from dopaminergic neurons in the ventral midbrain, governing behaviors such as anxiety or voluntary movement. To induce a pro-inflammatory environment, AgRP and POMC neuron-specific Gpx4 knockout mice were subjected to high-fat high-sucrose (HFHS) diet. To exacerbate oxidative stress in dopaminergic neurons of the ventral midbrain, we systemically co-deleted the PD-related gene DJ-1. Gpx4 was dispensable for the maintenance of cellular health and function of POMC neurons, even in mice exposed to obesogenic conditions. In contrast, HFHS-fed mice with Gpx4 deletion from AgRP neurons displayed increased body adiposity. Gpx4 expression and activity were diminished in the hypothalamus of HFHS-fed mice compared to standard diet-fed controls. Gpx4 deletion from dopaminergic neurons induced anxiety behavior, and diminished spontaneous locomotor activity when DJ-1 was co-deleted. Overall, these data suggest a physiological role for Gpx4 in balancing metabolic control signals and inflammation in AgRP but not POMC neurons. Moreover, Gpx4 appears to constitute an important rheostat against neuronal dysfunction and PD-like symptoms in dopaminergic circuitry within the ventral midbrain.

Glutathione peroxidase 4 inhibits Wnt/ß-catenin signaling and regulates dorsal organizer formation in zebrafish embryos.

The Wnt/ß-catenin signaling pathway plays pivotal roles in axis formation during embryogenesis and in adult tissue homeostasis. Glutathione peroxidase 4 (GPX4) is a selenoenzyme and participates in the reduction of peroxides. Its synthesis depends on the availability of the element selenium. However, the roles of GPX4 in vertebrate embryonic development and underlying mechanisms are largely unknown. Here, we show that maternal loss of zebrafish gpx4b promotes embryonic dorsal organizer formation, whereas overexpression of gpx4b inhibits the development of the dorsal organizer. Depletion of human GPX4 and zebrafish gpx4b (GPX4/gpx4b) increases, while GPX4/gpx4b overexpression decreases, Wnt/ß-catenin signaling in vivo and in vitro Functional and epistatic studies showed that GPX4 functions at the Tcf/Lef level, independently of selenocysteine activation. Mechanistically, GPX4 interacts with Tcf/Lefs and inhibits Wnt activity by preventing the binding of Tcf/Lefs to the promoters of Wnt target genes, resulting in inhibitory action in the presence of Wnt/ß-catenin signaling. Our findings unravel GPX4 as a suppressor of Wnt/ß-catenin signals, suggesting a possible relationship between the Wnt/ß-catenin pathway and selenium via the association of Tcf/Lef family proteins with GPX4.

ACSL4 dictates ferroptosis sensitivity by shaping cellular lipid composition.

Ferroptosis is a form of regulated necrotic cell death controlled by glutathione peroxidase 4 (GPX4). At present, mechanisms that could predict sensitivity and/or resistance and that may be exploited to modulate ferroptosis are needed. We applied two independent approaches-a genome-wide CRISPR-based genetic screen and microarray analysis of ferroptosis-resistant cell lines-to uncover acyl-CoA synthetase long-chain family member 4 (ACSL4) as an essential component for ferroptosis execution. Specifically, Gpx4-Acsl4 double-knockout cells showed marked resistance to ferroptosis. Mechanistically, ACSL4 enriched cellular membranes with long polyunsaturated ω6 fatty acids. Moreover, ACSL4 was preferentially expressed in a panel of basal-like breast cancer cell lines and predicted their sensitivity to ferroptosis. Pharmacological targeting of ACSL4 with thiazolidinediones, a class of antidiabetic compound, ameliorated tissue demise in a mouse model of ferroptosis, suggesting that ACSL4 inhibition is a viable therapeutic approach to preventing ferroptosis-related diseases.

Hepatocyte glutathione peroxidase-1 deficiency improves hepatic glucose metabolism and decreases steatohepatitis in mice.

AIMS/HYPOTHESIS: In obesity oxidative stress is thought to contribute to the development of insulin resistance, non-alcoholic fatty liver disease and the progression to non-alcoholic steatohepatitis. Our aim was to examine the precise contributions of hepatocyte-derived H2O2 to liver pathophysiology. METHODS: Glutathione peroxidase (GPX) 1 is an antioxidant enzyme that is abundant in the liver and converts H2O2 to water. We generated Gpx1 lox/lox mice to conditionally delete Gpx1 in hepatocytes (Alb-Cre;Gpx1 lox/lox) and characterised mice fed chow, high-fat or choline-deficient amino-acid-defined (CDAA) diets. RESULTS: Chow-fed Alb-Cre;Gpx1 lox/lox mice did not exhibit any alterations in body composition or energy expenditure, but had improved insulin sensitivity and reduced fasting blood glucose. This was accompanied by decreased gluconeogenic and increased glycolytic gene expression as well as increased hepatic glycogen. Hepatic insulin receptor Y1163/Y1163 phosphorylation and Akt Ser-473 phosphorylation were increased in fasted chow-fed Alb-Cre;Gpx1 lox/lox mice, associated with increased H2O2 production and insulin signalling in isolated hepatocytes. The enhanced insulin signalling was accompanied by the increased oxidation of hepatic protein tyrosine phosphatases previously implicated in the attenuation of insulin signalling. High-fat-fed Alb-Cre;Gpx1 lox/lox mice did not exhibit alterations in weight gain or hepatosteatosis, but exhibited decreased hepatic inflammation, decreased gluconeogenic gene expression and increased insulin signalling in the liver. Alb-Cre;Gpx1 lox/lox mice fed a CDAA diet that promotes non-alcoholic steatohepatitis exhibited decreased hepatic lymphocytic infiltrates, inflammation and liver fibrosis. CONCLUSIONS/INTERPRETATION: Increased hepatocyte-derived H2O2 enhances hepatic insulin signalling, improves glucose control and protects mice from the development of non-alcoholic steatohepatitis.

Genetic depletion of glutathione peroxidase-1 potentiates nephrotoxicity induced by multiple doses of cocaine via activation of angiotensin II AT1 receptor.

We investigated the possible roles of angiotensin II type 1 receptor (AT1R) and oxidative stress responsive nuclear factor κB (NFκB) in renal damage caused by multiple doses of cocaine in glutathione peroxidase (GPx)-1 gene-depleted mice. Treatment with cocaine resulted in significant increases in malondialdehyde, protein carbonyl, and pro-apoptotic Bax expression and decreases in the ratio of glutathione (GSH) and its oxidized form (GSSG), GSH-dependent enzymes, and anti-apoptotic factors in the kidney. These alterations were more pronounced in GPx-1 knockout (-/-) mice than in wild type (WT) mice. Notably, the AT1R antagonist losartan protected against the renal toxicity induced by cocaine, whereas the NFκB inhibitor pyrrolidine dithiocarbamate was not protective. The toxicity was more pronounced in GPx-1 (-/-) mice than in WT mice. The protective effect afforded by losartan against cocaine toxicity appeared to be more sensitive in GPx-1 (-/-) mice than that in WT mice. These losartan-mediated protective effects were inhibited by the phosphatidyl-inositol-3-kinase (PI3K) inhibitor LY294002, indicating that losartan provides significant protection from cocaine-induced renal toxicity through PI3K/Akt signaling. Our results suggest that genetic inhibition of GPx-1 potentiates cocaine-induced renal damage via activation of AT1R by inhibition of PI3K-Akt signaling, and that AT1R can be a therapeutic target against renal toxicity induced by cocaine.

Lack of glutathione peroxidase-1 facilitates a pro-inflammatory and activated vascular endothelium.

A critical early event in the pathogenesis of atherosclerosis is vascular inflammation leading to endothelial dysfunction (ED). Reactive oxygen species and inflammation are inextricably linked and declining antioxidant defense is implicated in ED. We have previously shown that Glutathione peroxidase-1 (GPx1) is a crucial antioxidant enzyme in the protection against diabetes-associated atherosclerosis. In this study we aimed to investigate mechanisms by which lack of GPx1 affects pro-inflammatory mediators in primary aortic endothelial cells (PAECs) isolated from GPx1 knockout (GPx1 KO) mice. Herein, we demonstrate that lack of GPx1 prolonged TNF-α induced phosphorylation of P38, ERK and JNK, all of which was reversed upon treatment with the GPx1 mimetic, ebselen. In addition, Akt phosphorylation was reduced in GPx1 KO PAECs, which correlated with decreased nitric oxide (NO) bioavailability as compared to WT PAECs. Furthermore, IκB degradation was prolonged in GPx1 KO PAECS suggesting an augmentation of NF-κB activity. In addition, the expression of vascular cell adhesion molecule (VCAM-1) was significantly increased in GPx1 KO PAECs and aortas. Static and dynamic flow adhesion assays showed significantly increased adhesion of fluorescently labeled leukocytes to GPx1 KO PAECS and aortas respectively, which were significantly reduced by ebselen treatment. Our results suggest that GPx1 plays a critical role in regulating pro-inflammatory pathways, including MAPK and NF-κB, and down-stream mediators such as VCAM-1, in vascular endothelial cells. Lack of GPx1, via effects on p-AKT also affects signaling to eNOS-derived NO. We speculate based on these results that declining antioxidant defenses as seen in cardiovascular diseases, by failing to regulate these pro-inflammatory pathways, facilitates an inflammatory and activated endothelium leading to ED and atherogenesis.

Plasmalogen modulation attenuates atherosclerosis in ApoE- and ApoE/GPx1-deficient mice.

BACKGROUND AND AIM: We previously reported a negative association of circulating plasmalogens (phospholipids with proposed atheroprotective properties) with coronary artery disease. Plasmalogen modulation was previously demonstrated in animals but its effect on atherosclerosis was unknown. We assessed the effect of plasmalogen enrichment on atherosclerosis of murine models with differing levels of oxidative stress. METHODS AND RESULTS: Six-week old ApoE- and ApoE/glutathione peroxidase-1 (GPx1)-deficient mice were fed a high-fat diet with/without 2% batyl alcohol (precursor to plasmalogen synthesis) for 12 weeks. Mass spectrometry analysis of lipids showed that batyl alcohol supplementation to ApoE- and ApoE/GPx1-deficient mice increased the total plasmalogen levels in both plasma and heart. Oxidation of plasmalogen in the treated mice was evident from increased level of plasmalogen oxidative by-product, sn-2 lysophospholipids. Atherosclerotic plaque in the aorta was reduced by 70% (P = 5.69E-07) and 69% (P = 2.00E-04) in treated ApoE- and ApoE/GPx1-deficient mice, respectively. A 40% reduction in plaque (P = 7.74E-03) was also seen in the aortic sinus of only the treated ApoE/GPx1-deficient mice. Only the treated ApoE/GPx1-deficient mice showed a decrease in VCAM-1 staining (-28%, P = 2.43E-02) in the aortic sinus and nitrotyrosine staining (-78%, P = 5.11E-06) in the aorta. CONCLUSION: Plasmalogen enrichment via batyl alcohol supplementation attenuated atherosclerosis in ApoE- and ApoE/GPx1-deficient mice, with a greater effect in the latter group. Plasmalogen enrichment may represent a viable therapeutic strategy to prevent atherosclerosis and reduce cardiovascular disease risk, particularly under conditions of elevated oxidative stress and inflammation.

Genome-wide hydroxymethylcytosine pattern changes in response to oxidative stress.

The TET enzymes convert methylcytosine to the newly discovered base hydroxymethylcytosine. While recent reports suggest that TETs may play a role in response to oxidative stress, this role remains uncertain, and results lack in vivo models. Here we show a global decrease of hydroxymethylcytosine in cells treated with buthionine sulfoximine, and in mice depleted for the major antioxidant enzymes GPx1 and 2. Furthermore, genome-wide profiling revealed differentially hydroxymethylated regions in coding genes, and intriguingly in microRNA genes, both involved in response to oxidative stress. These results thus suggest a profound effect of in vivo oxidative stress on the global hydroxymethylome.

The effect of alcohol and hydrogen peroxide on liver hepcidin gene expression in mice lacking antioxidant enzymes, glutathione peroxidase-1 or catalase.

This study investigates the regulation of hepcidin, the key iron-regulatory molecule, by alcohol and hydrogen peroxide (H2O2) in glutathione peroxidase-1 (gpx-1(-/-)) and catalase (catalase(-/-)) knockout mice. For alcohol studies, 10% ethanol was administered in the drinking water for 7 days. Gpx-1(-/-) displayed significantly higher hepatic H2O2 levels than catalase(-/-) compared to wild-type mice, as measured by 2'-7'-dichlorodihydrofluorescein diacetate (DCFH-DA). The basal level of liver hepcidin expression was attenuated in gpx-1(-/-) mice. Alcohol increased H2O2 production in catalase(-/-) and wild-type, but not gpx-1(-/-), mice. Hepcidin expression was inhibited in alcohol-fed catalase(-/-) and wild-type mice. In contrast, alcohol elevated hepcidin expression in gpx-1(-/-) mice. Gpx-1(-/-) mice also displayed higher level of basal liver CHOP protein expression than catalase(-/-) mice. Alcohol induced CHOP and to a lesser extent GRP78/BiP expression, but not XBP1 splicing or binding of CREBH to hepcidin gene promoter, in gpx-1(-/-) mice. The up-regulation of hepatic ATF4 mRNA levels, which was observed in gpx-1(-/-) mice, was attenuated by alcohol. In conclusion, our findings strongly suggest that H2O2 inhibits hepcidin expression in vivo. Synergistic induction of CHOP by alcohol and H2O2, in the absence of gpx-1, stimulates liver hepcidin gene expression by ER stress independent of CREBH.

Silencing GPX3 Expression Promotes Tumor Metastasis in Human Thyroid Cancer.

The incidence of thyroid cancer has been increased in the past few decades. GPX3 gene is located in 5q23, which is frequently deleted in prostate cancer. Methylation of GPX3 was found frequently in prostate, esophagus, gastric and breast cancers. To detect the expression and analyze the mechanism of GPX3 in human thyroid cancer, 94 cases of primary papillary thyroid cancer (PTC) which coupled with adjacent tissue samples, as well as 15 cases of normal thyroid epithelial tissue samples were collected. Methylation specific PCR (MSP), immunohistochemistry staining, transwell assay and siRNA knockdown technique were employed. GPX3 was methylated in 46.8% (44 of 94) of PTC and methylation of GPX3 was associated with tumor size (P<0.05) and regional lymph node metastasis (P<0.01) significantly. Consistent expression of GPX3 was observed in the adjacent tissue samples and absent/reduced expression of GPX3 was found frequently in primary PTC samples. In 44 cases of methylated cancer samples, loss of/ reduced expression was found in 36 cases and expression of GPX3 was found in 8 cases. In 50 cases of unmethylated PTC samples, loss of/ reduced expression was found in 31 cases and expression of GPX3 was found in 19 cases. Lost/ reduced expression of GPX3 is associated with promoter region hypermethylation (P<0.05). Wnt signaling was inhibited by GPX3 in TPC-1 and FTC133 cells. In conclusion, GPX3 is frequently methylated in human papillary thyroid cancer and the expression of GPX3 was regulated by promoter region methylation. Methylation of GPX3 is related to tumor size and lymph node metastasis. Metastasis of thyroid cancer was suppressed by GPX3 through inhibition Wnt/ß-catenin signaling.

Hepatic oxidative stress promotes insulin-STAT-5 signaling and obesity by inactivating protein tyrosine phosphatase N2.

Hepatic insulin resistance is a key contributor to the pathogenesis of obesity and type 2 diabetes (T2D). Paradoxically, the development of insulin resistance in the liver is not universal, but pathway selective, such that insulin fails to suppress gluconeogenesis but promotes lipogenesis, contributing to the hyperglycemia, steatosis, and hypertriglyceridemia that underpin the deteriorating glucose control and microvascular complications in T2D. The molecular basis for the pathway-specific insulin resistance remains unknown. Here we report that oxidative stress accompanying obesity inactivates protein-tyrosine phosphatases (PTPs) in the liver to activate select signaling pathways that exacerbate disease progression. In obese mice, hepatic PTPN2 (TCPTP) inactivation promoted lipogenesis and steatosis and insulin-STAT-5 signaling. The enhanced STAT-5 signaling increased hepatic IGF-1 production, which suppressed central growth hormone release and exacerbated the development of obesity and T2D. Our studies define a mechanism for the development of selective insulin resistance with wide-ranging implications for diseases characterized by oxidative stress.