Absence of glutathione peroxidase-1 exacerbates cerebral ischemia-reperfusion injury by reducing post-ischemic microvascular perfusion

Mice deficient in the anti-oxidant enzyme glutathione peroxidase-1 (Gpx1) have a greater susceptibility to cerebral injury following a localized ischemic event. Much of the response to ischemia-reperfusion is caused by aberrant responses within the microvasculature, including inflammation, diminished endothelial barrier function (increased vascular permeability), endothelial activation, and reduced microvascular perfusion. However, the role of Gpx1 in regulating these responses has not been investigated. Wild-type and Gpx1-/- mice underwent focal cerebral ischemia via mid-cerebral artery occlusion followed by measurement of cerebral perfusion via laser Doppler and intravital microscopy. Post-ischemic brains in wild-type mice displayed significant deficit in microvascular perfusion. However, in Gpx1-/- mice, the deficit in cerebral blood flow was significantly greater than that in wild-type mice, and this was associated with significant increase in infarct size and increased vascular permeability. Ischemia-reperfusion also resulted in expression of matrix metalloproteinase-9 (MMP-9) in endothelial cells. The absence of Gpx1 was associated with marked increase in pro-MMP-9 expression as well as potentiated MMP-9 activity. Pre-treatment of Gpx1-/- mice with the anti-oxidant ebselen restored microvascular perfusion, limited the induction and activation of MMP-9, and attenuated the increases in infarct size and vascular permeability. These findings demonstrate that the anti-oxidant function of Gpx1 plays a critical role in protecting the cerebral microvasculature against ischemia-reperfusion injury by preserving microvascular perfusion and inhibiting MMP-9 expression.     

Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene

Oxidative stress resulting from mitochondrially derived reactive oxygen species (ROS) has been hypothesized to damage mitochondrial oxidative phosphorylation (OXPHOS) and to be a factor in aging and degenerative disease. If this hypothesis is correct, then genetically inactivating potential mitochondrial antioxidant enzymes such as glutathione peroxidase-1 (Gpx1; EC 1.11.1.9) should increase mitochondrial ROS production and decrease OXPHOS function. To determine the expression pattern of Gpx1, isoform-specific antibodies were generated and mutant mice were prepared in which the Gpx1 protein was substituted for by beta-galactosidase, driven by the Gpx1 promoter. These experiments revealed that Gpx1 is highly expressed in both the mitochondria and the cytosol of the liver and kidney, but poorly expressed in heart and muscle. To determine the physiological importance of Gpx1, mice lacking Gpx1 were generated by targeted mutagenesis in mouse ES cells. Homozygous mutant Gpx1(tm1Mgr) mice have 20% less body weight than normal animals and increased levels of lipid peroxides in the liver. Moreover, the liver mitochondria were found to release markedly increased hydrogen peroxide, a Gpx1 substrate, and have decreased mitochondrial respiratory control ratio and power output index. Hence, genetic inactivation of Gpx1 resulted in growth retardation, presumably due in part to reduced mitochondrial energy production as a product of increased oxidative stress.     

Nox1 causes ileocolitis in mice deficient in glutathione peroxidase-1 and -2

We previously reported that mice deficient in two Se-dependent glutathione peroxidases, GPx1 and GPx2, have spontaneous ileocolitis. Disease severity depends on mouse genetic background. Whereas C57BL/6J (B6) GPx1/2-double-knockout (DKO) mice have moderate ileitis and mild colitis, 129S1Svlm/J (1 2 9) DKO mice have severe ileocolitis. Because GPx’s are antioxidant enzymes, we hypothesized that elevated reactive oxygen species trigger inflammation in these DKO mice. To test whether NADPH oxidase 1 (Nox1) contributes to colitis, we generated B6 triple-KO (TKO) mice to study their phenotype. Because the Nox1 gene is X-linked, we analyzed the effects of Nox1 on male B6 TKO mice and female B6 DKO mice with the Nox1^+/− (het-TKO) genotype. We found that the male TKO and female het-TKO mice are virtually disease-free when monitored from 8 through 50 days of age. Male TKO and female het-TKO mice have nearly no signs of disease (e.g., lethargy and perianal alopecia) that are often exhibited in the DKO mice; further, the slower growth rate of DKO mice is almost completely eliminated in male TKO and female het-TKO mice. Male TKO and female het-TKO mice no longer have the shortened small intestine present in the DKO mice. Finally, the pathological characteristics of the DKO ileum, including the high level of crypt apoptosis (analyzed by apoptotic figures, TUNEL, and cleaved caspase-3 immunohistochemical staining), high numbers of Ki-67-positive crypt epithelium cells, and elevated levels of monocytes expressing myeloperoxidase, are all significantly decreased in male TKO mice. The attenuated ileal and colonic pathology is also evident in female het-DKO mice. Furthermore, the male DKO ileum has eightfold higher TNF cytokine levels than TKO ileum. Nox1 mRNA is highly elevated in both B6 and 129 DKO ileum compared to wild-type mouse ileum. Taking these results together, we propose that ileocolitis in the DKO mice is caused by Nox1, which is induced by TNF. The milder disease in female het-TKO intestine is probably due to random or imprinted X-chromosome inactivation, which produces mosaic Nox1 expression.     

A multifaceted view on the impacts of shrub encroachment

Shrub encroachment, a global phenomenon with management implications, is examined in two papers in the current issue of Applied Vegetation Science. Barbosa da Silva et al. show that encroachment simplifies the herbaceous community, and Pittarello et al. illustrate how pastoral practices can restore encroached grasslands. While detrimental effects of shrub encroachment on grassland vegetation are often reported, we argue for a more holistic view when assessing this land‐cover change.     

Increased infarct size and exacerbated apoptosis in the glutathione peroxidase-1 (Gpx-1) knockout mouse brain in response to ischemia/reperfusion injury

Glutathione peroxidase is an antioxidant enzyme that is involved in the control of cellular oxidative state. Recently, unregulated oxidative state has been implicated as detrimental to neural cell viability and involved in both acute and chronic neurodegeneration. In this study we have addressed the importance of a functional glutathione peroxidase in a mouse ischemia/reperfusion model. Two hours of focal cerebral ischemia followed by 24 h of reperfusion was induced via the intraluminal suture method. Infarct volume was increased three-fold in the glutathione peroxidase-1 (Gpx-1) -/- mouse compared with the wild-type mouse; this was mirrored by an increase in the level of apoptosis found at 24 h in the Gpx-1 -/- mouse compared with the wild-type mouse. Neuronal deficit scores correlated to the histologic data. We also found that activated caspase-3 expression is present at an earlier time point in the Gpx-1 -/- mice when compared with the wild-type mice, which suggests an enhanced susceptibility to apoptosis in the Gpx-1 -/- mouse. This is the first known report of such a dramatic increase, both temporally and in level of apoptosis in a mouse stroke model. Our results suggest that Gpx-1 plays an important regulatory role in the protection of neural cells in response to the extreme oxidative stress that is released during ischemia/reperfusion injury.     

Analysis of candidate colitis genes in the gdac1 locus of mice deficient in glutathione peroxidase-1 and -2

BACKGROUND: Mice that are deficient for glutathione peroxidases 1 and 2 (GPX) show large variations in the penetrance and severity of colitis in C57BL/6J and 129S1/SvImJ backgrounds. We mapped a locus contributing to this difference to distal chromosome 2 (～119-133 mbp) and named it glutathione peroxidase-deficiency-associated colitis 1 (Gdac1). The aim of this study was to identify the best gene candidates within the Gdac1 locus contributing to the murine colitis phenotype. METHOD/PRINCIPAL FINDINGS: We refined the boundaries of Gdac1 to 118-125 mbp (95% confidence interval) by increasing sample size and marker density across the interval. The narrowed region contains 128 well-annotated protein coding genes but it excludes Fermt1, a human inflammatory bowel disease candidate that was within the original boundaries of Gdac1. The locus we identified may be the Cdcs3 locus mapped by others studying IL10-knockout mice. Using in silico analysis of the 128 genes, based on published colon expression data, the relevance of pathways to colitis, gene mutations, presence of non-synonymous-single-nucleotide polymorphisms (nsSNPs) and whether the nsSNPs are predicted to have an impact on protein function or expression, we excluded 42 genes. Based on a similar analysis, twenty-five genes from the remaining 86 genes were analyzed for expression-quantitative-trait loci, and another 15 genes were excluded. CONCLUSION/SIGNIFICANCE: Among the remaining 10 genes, we identified Pla2g4f and Duox2 as the most likely colitis gene candidates, because GPX metabolizes PLA2G4F and DUOX2 products. Pla2g4f is a phospholipase A2 that has three potentially significant nsSNP variants and showed expression differences across mouse strains. PLA2G4F produces arachidonic acid, which is a substrate for lipoxygenases and, in turn, for GPXs. DUOX2 produces H(2)O(2) and may control microbial populations. DUOX-1 and -2 control microbial populations in mammalian lung and in the gut of several insects and zebrafish. Dysbiosis is a phenotype that differentiates 129S1/SvImJ from C57BL/6J and may be due to strain differences in DUOX2 activity.     

Cis-acting elements are required for selenium regulation of glutathione peroxidase-1 mRNA levels.

Classical glutathione peroxidase (GPX1) mRNA levels can decrease to less than 10% in selenium (Se)-deficient rat liver. The cis-acting nucleic acid sequence requirements for Se regulation of GPX1 mRNA levels were studied by transfecting Chinese hamster ovary (CHO) cells with GPX1 DNA constructs in which specific regions of the GPX1 gene were mutated, deleted, or replaced by comparable regions from unregulated genes such as phospholipid hydroperoxide glutathione peroxidase (GPX4). For each construct, stable transfectants were pooled two weeks after transfection, divided into Se-deficient (2 nM Se) or Se-adequate (200 nM Se) medium, and grown for an additional four days. On day of harvest, Se-deficient GPX1 and GPX4 activities averaged 13 +/- 2% and 15 +/- 2% of Se adequate levels, confirming that cellular Se status was dramatically altered by Se supplementation. RNA was isolated from replicate plates of cells and transfected mRNA levels were specifically determined by RNase protection assay. Analysis of chimeric GPX1/GPX4 constructs showed that the GPX4 3'-UTR can completely replace the GPX1 3'-UTR in Se regulation of GPX1 mRNA. We did not find any GPX1 coding regions that could be replaced by the corresponding GPX4 coding regions without diminishing or eliminating Se regulation of the transfected GPX1 mRNA. Further analysis of the GPX1 coding region demonstrated that the GPX1 Sec codon (UGA) and the GPX1 intron sequences are required for full Se regulation of transfected GPX1 mRNA levels. Mutations that moved the GPX1 Sec codon to three different positions within the GPX1 coding region suggest that the mechanism for Se regulation of GPX1 mRNA requires a Sec codon within exon 1. Lastly, we found that addition of the GPX1 3'-UTR to beta-globin mRNA can convey significant Se regulation to beta-globin mRNA levels when a UGA codon is placed within exon 1. We conclude that Se regulation of GPX1 mRNA requires a functional selenocysteine insertion sequence (SECIS) in the 3'-UTR and a Sec codon followed by an intron.     

Opposite roles of selenium-dependent glutathione peroxidase-1 in superoxide generator diquat- and peroxynitrite-induced apoptosis and signaling

Oxidative injuries including apoptosis can be induced by reactive oxygen species (ROS) and reactive nitrogen species (RNS) in aerobic metabolism. We determined impacts of a selenium-dependent glutathione peroxidase-1 (GPX1) on apoptosis induced by diquat (DQ), a ROS (superoxide) generator, and peroxynitrite (PN), a potent RNS. Hepatocytes were isolated from GPX1 knockout (GPX1-/-) or wild-type (WT) mice, and treated with 0.5 mm DQ or 0.1-0.8 mm PN for up to 12 h. Loss of cell viability, high levels of apoptotic cells, and severe DNA fragmentation were produced by DQ in only GPX1-/- cells and by PN in only WT cells. These two groups of cells shared similar cytochrome c release, caspase-3 activation, and p21(WAF1/CIP1) cleavage. Higher levels of protein nitration were induced by PN in WT than GPX1-/- cells. Much less and/or slower cellular GSH depletion was caused by DQ or PN in GPX1-/- than in WT cells, and corresponding GSSG accumulation occurred only in the latter. In conclusion, it is most striking that, although GPX1 protects against apoptosis induced by superoxide-generator DQ, the enzyme actually promotes apoptosis induced by PN in murine hepatocytes. Indeed, GSH is a physiological substrate for GPX1 in coping with ROS in these cells.     

A multifaceted role for ATM in genome maintenance

The pleiotropic nature of the clinical phenotypes of patients with ataxia-telangiectasia (A-T)--which encompass cerebellar degeneration (leading to ataxia), gonadal atrophy, and cancer predisposition--suggests multiple functions of the gene responsible for the disease. The ataxia-telangiectasia mutated gene product (ATM), whose loss of function is responsible for ataxia-telangiectasia, is a protein kinase that interacts with several substrates and is implicated in mitogenic signal transduction, chromosome condensation, meiotic recombination, cell-cycle control and telomere maintenance. This review focuses on the critical roles that ATM appears to play in cell-cycle checkpoints, DNA repair, telomere metabolism and oxidative stress, indicating how defects in these processes might lead to ataxia-telangiectasia.     

The multifaceted role of mTORC1 in the control of lipid metabolism

The mechanistic target of rapamycin is a protein kinase that, as part of the mechanistic target of rapamycin complex 1 (mTORC1), senses both local nutrients and, through insulin signalling, systemic nutrients to control a myriad of cellular processes. Although roles for mTORC1 in promoting protein synthesis and inhibiting autophagy in response to nutrients have been well established, it is emerging as a central regulator of lipid homeostasis. Here, we discuss the growing genetic and pharmacological evidence demonstrating the functional importance of its signalling in controlling mammalian lipid metabolism, including lipid synthesis, oxidation, transport, storage and lipolysis, as well as adipocyte differentiation and function. Defining the role of mTORC1 signalling in these metabolic processes is crucial to understanding the pathophysiology of obesity and its relationship to complex diseases, including diabetes and cancer.     

The multifaceted role of TMEM16A in cancer

The calcium-activated chloride channel TMEM16A is intimately linked to cancers. Over decades, TMEM16A over-expression and contribution to prognosis have been widely studied for multiple cancers strengthening the idea that TMEM16A could be a valuable biomarker and a promising therapeutic target. Surprisingly, from the survey of the literature, it appears that TMEM16A has been involved in multiple cancer-related functions and a large number of molecular targets of TMEM16A have been proposed. Thus, TMEM16A appears to be an ion channel with a multifaceted role in cancers.In this review, we summarize the latest development regarding TMEM16A contribution to cancers. We will survey TMEM16A contribution in cancer prognosis, the origins of its over-expression in cancer cells, the multiple biological functions and molecular pathways regulated by TMEM16A. Then, we will consider the question regarding the molecular mechanism of TMEM16A in cancers and the possible basis for the multifaceted role of TMEM16A in cancers.     

A global view of antibiotic resistance

Antibiotic resistance is one of the few examples of evolution that can be addressed experimentally. The present review analyses this resistance, focusing on the networks that regulate its acquisition and its effect on bacterial physiology. It is widely accepted that antibiotics and antibiotic resistance genes play fundamental ecological roles – as weapons and shields, respectively – in shaping the structures of microbial communities. Although this Darwinian view of the role of antibiotics is still valid, recent work indicates that antibiotics and resistance mechanisms may play other ecological roles and strongly influence bacterial physiology. The expression of antibiotic resistance determinants must therefore be tightly regulated and their activity forms part of global metabolic networks. In addition, certain bacterial modes of life can trigger transient phenotypic antibiotic resistance under some circumstances. Understanding resistance thus requires the analysis of the regulatory networks controlling bacterial evolvability, the physiological webs affected and the metabolic rewiring it incurs.     

Suppression of atherogenesis by overexpression of glutathione peroxidase-4 in apolipoprotein E-deficient mice

Accumulation of oxidized lipids in the arterial wall contributes to atherosclerosis. Glutathione peroxidase-4 (GPx4) is a hydroperoxide scavenger that removes oxidative modifications from lipids such as free fatty acids, cholesterols, and phospholipids. Here, we set out to assess the effects of GPx4 overexpression on atherosclerosis in apolipoprotein E-deficient (ApoE(-/-)) mice. The results revealed that atherosclerotic lesions in the aortic tree and aortic sinus of ApoE(-/-) mice overexpressing GPx4 (hGPx4Tg/ApoE(-/-)) were significantly smaller than those of ApoE(-/-) control mice. GPx4 overexpression also diminished signs of advanced lesions in the aortic sinus, as seen by a decreased occurrence of fibrous caps and acellular areas among hGPx4Tg/ApoE(-/-) animals. This delay of atherosclerosis in hGPx4Tg/ApoE(-/-) mice correlated with reduced aortic F(2)-isoprostane levels (R(2)=0.75, p<0.01). In addition, overexpression of GPx4 lessened atherogenic events induced by the oxidized lipids lysophosphatidylcholine and 7-ketocholesterol, including upregulated expression of adhesion molecules in endothelial cells and adhesion of monocytes to endothelial cells, as well as endothelial necrosis and apoptosis. These results suggest that overexpression of GPx4 inhibits the development of atherosclerosis by decreasing lipid peroxidation and inhibiting the sensitivity of vascular cells to oxidized lipids.     

The role of glutathione in the isomerization of delta 5-androstene-3,17-dione catalyzed by human glutathione transferase A1-1

Human glutathione transferase (GST) A1-1 efficiently catalyzes the isomerization of Delta(5)-androstene-3,17-dione (AD) into Delta(4)-androstene-3,17-dione. High activity requires glutathione, but enzymatic catalysis occurs also in the absence of this cofactor. Glutathione alone shows a limited catalytic effect. S-Alkylglutathione derivatives do not promote the reaction, and the pH dependence of the isomerization indicates that the glutathione thiolate serves as a base in the catalytic mechanism. Mutation of the active-site Tyr(9) into Phe significantly decreases the steady-state kinetic parameters, alters their pH dependence, and increases the pK(a) value of the enzyme-bound glutathione thiol. Thus, Tyr(9) promotes the reaction via its phenolic hydroxyl group in protonated form. GST A2-2 has a catalytic efficiency with AD 100-fold lower than the homologous GST A1-1. Another Alpha class enzyme, GST A4-4, is 1000-fold less active than GST A1-1. The Y9F mutant of GST A1-1 is more efficient than GST A2-2 and GST A4-4, both having a glutathione cofactor and an active-site Tyr(9) residue. The active sites of GST A2-2 and GST A1-1 differ by only four amino acid residues, suggesting that proper orientation of AD in relation to the thiolate of glutathione is crucial for high catalytic efficiency in the isomerization reaction. The GST A1-1-catalyzed steroid isomerization provides a complement to the previously described isomerase activity of 3beta-hydroxysteroid dehydrogenase.     

The multifaceted role of plasminogen in inflammation

A fine-tuned activation and deactivation of proteases and their inhibitors are involved in the execution of the inflammatory response. The zymogen/proenzyme plasminogen is converted to the serine protease plasmin, a key fibrinolytic factor by plasminogen activators including tissue-type plasminogen activator (tPA). Plasmin is part of an intricate protease network controlling proteins of initial hemostasis/coagulation, fibrinolytic and complement system. Activation of these protease cascades is required to mount a proper inflammatory response. Although best known for its ability to dissolve clots and cleave fibrin, recent studies point to the importance of fibrin-independent functions of plasmin during acute inflammation and inflammation resolution. In this review, we provide an up-to-date overview of the current knowledge of the enzymatic and cytokine-like effects of tPA and describe the role of tPA and plasminogen receptors in the regulation of the inflammatory response with emphasis on the cytokine storm syndrome such as observed during coronavirus disease 2019 or macrophage activation syndrome. We discuss tPA as a modulator of Toll like receptor signaling, plasmin as an activator of NFkB signaling, and summarize recent studies on the role of plasminogen receptors as controllers of the macrophage conversion into the M2 type and as mediators of efferocytosis during inflammation resolution.