Glutathione peroxidase 4 and vitamin E cooperatively prevent hepatocellular degeneration.
ABSTRACT: The selenoenzyme glutathione peroxidase 4 (Gpx4) is an essential mammalian glutathione peroxidase, which protects cells against detrimental lipid peroxidation and governs a novel form of regulated necrotic cell death, called ferroptosis. To study the relevance of Gpx4 and of another vitally important selenoprotein, cytosolic thioredoxin reductase (Txnrd1), for liver function, mice with conditional deletion of Gpx4 in hepatocytes were studied, along with those lacking Txnrd1 and selenocysteine (Sec) tRNA (Trsp) in hepatocytes. Unlike Txnrd1- and Trsp-deficient mice, Gpx4-/- mice died shortly after birth and presented extensive hepatocyte degeneration. Similar to Txnrd1-deficient livers, Gpx4-/- livers manifested upregulation of nuclear factor (erythroid-derived)-like 2 (Nrf2) response genes. Remarkably, Gpx4-/- pups born from mothers fed a vitamin E-enriched diet survived, yet this protection was reversible as subsequent vitamin E deprivation caused death of Gpx4-deficient mice ~4 weeks thereafter. Abrogation of selenoprotein expression in Gpx4-/- mice did not result in viable mice, indicating that the combined deficiency aggravated the loss of Gpx4 in liver. By contrast, combined Trsp/Txnrd1-deficient mice were born, but had significantly shorter lifespans than either single knockout, suggesting that Txnrd1 plays an important role in supporting liver function of mice lacking Trsp. In sum our study demonstrates that the ferroptosis regulator Gpx4 is critical for hepatocyte survival and proper liver function, and that vitamin E can compensate for its loss by protecting cells against deleterious lipid peroxidation.
Project description:Changes in dietary selenium and selenoprotein status may influence both anti- and pro-cancer pathways, making the outcome of interventions different from one study to another. To characterize such outcomes in a defined setting, we undertook a controlled hepatocarcinogenesis study involving varying levels of dietary selenium and altered selenoprotein status using mice carrying a mutant (A37G) selenocysteine tRNA transgene (Trsp(tG37) ) and/or a cancer driver TGF? transgene. The use of Trsp(tG37) altered selenoprotein expression in a selenoprotein and tissue specific manner and, at sufficient dietary selenium levels, separate the effect of diet and selenoprotein status. Mice were maintained on diets deficient in selenium (0.02 ppm selenium) or supplemented with 0.1, 0.4 or 2.25 ppm selenium or 30 ppm triphenylselenonium chloride (TPSC), a non-metabolized selenium compound. Trsp(tG37) transgenic and TGF?/Trsp(tG37) bi-transgenic mice subjected to selenium-deficient or TPSC diets developed a neurological phenotype associated with early morbidity and mortality prior to hepatocarcinoma development. Pathology analyses revealed widespread disseminated pyogranulomatous inflammation. Pyogranulomas occurred in liver, lungs, heart, spleen, small and large intestine, and mesenteric lymph nodes in these transgenic and bi-transgenic mice. The incidence of liver tumors was significantly increased in mice carrying the TGF? transgene, while dietary selenium and selenoprotein status did not affect tumor number and multiplicity. However, adenoma and carcinoma size and area were smaller in TGF? transgenic mice that were fed 0.4 and 2.25 versus 0.1 ppm of selenium. Thus, selenium and selenoprotein deficiencies led to widespread pyogranuloma formation, while high selenium levels inhibited the size of TGF?-induced liver tumors.
Project description:Glutathione peroxidase 4 (GPX4), an antioxidant defense enzyme active in repairing oxidative damage to lipids, is a key inhibitor of ferroptosis, a non-apoptotic form of cell death involving lipid reactive oxygen species. Here we show that GPX4 is essential for motor neuron health and survival in vivo. Conditional ablation of Gpx4 in neurons of adult mice resulted in rapid onset and progression of paralysis and death. Pathological inspection revealed that the paralyzed mice had a dramatic degeneration of motor neurons in the spinal cord but had no overt neuron degeneration in the cerebral cortex. Consistent with the role of GPX4 as a ferroptosis inhibitor, spinal motor neuron degeneration induced by Gpx4 ablation exhibited features of ferroptosis, including no caspase-3 activation, no TUNEL staining, activation of ERKs, and elevated spinal inflammation. Supplementation with vitamin E, another inhibitor of ferroptosis, delayed the onset of paralysis and death induced by Gpx4 ablation. Also, lipid peroxidation and mitochondrial dysfunction appeared to be involved in ferroptosis of motor neurons induced by Gpx4 ablation. Taken together, the dramatic motor neuron degeneration and paralysis induced by Gpx4 ablation suggest that ferroptosis inhibition by GPX4 is essential for motor neuron health and survival in vivo.
Project description:Ferroptosis is a necrotic form of regulated cell death (RCD) mediated by phospholipid peroxidation in association with free iron-mediated Fenton reactions. Disrupted iron homeostasis resulting in excessive oxidative stress has been implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, we demonstrate the involvement of ferroptosis in COPD pathogenesis. Our in vivo and in vitro models show labile iron accumulation and enhanced lipid peroxidation with concomitant non-apoptotic cell death during cigarette smoke (CS) exposure, which are negatively regulated by GPx4 activity. Treatment with deferoxamine and ferrostatin-1, in addition to GPx4 knockdown, illuminate the role of ferroptosis in CS-treated lung epithelial cells. NCOA4-mediated ferritin selective autophagy (ferritinophagy) is initiated during ferritin degradation in response to CS treatment. CS exposure models, using both GPx4-deficient and overexpressing mice, clarify the pivotal role of GPx4-regulated cell death during COPD. These findings support a role for cigarette smoke-induced ferroptosis in the pathogenesis of COPD.
Project description:Although various lines of evidence suggest that oxidative stress plays a role in human prostate cancer initiation and progression, there is a paucity of direct evidence for its role in tumor initiation. To begin to address this issue, we developed a novel tumorigenesis model by reducing the expression of multiple selenoproteins (SPs) in mouse prostatic epithelium. This was accomplished via the prostate-specific deletion of Trsp, a gene that encodes a transfer RNA (Sec tRNA) required for the insertion of selenocysteine residues into SPs during their translation. By 6 weeks of age, Trsp-deficient mice exhibited widespread prostatic intraepithelial neoplasia lesions in all prostatic lobes, which then progressed to high-grade dysplasia and microinvasive carcinoma by 24 weeks. In contrast to other murine prostate cancer models, Trsp-deficient mice required neither the deletion of a tumor suppressor nor the transgenic introduction of an oncogene for prostatic intraepithelial neoplasia lesion development. In keeping with the antioxidant functions of several SPs, we found increases in lipid peroxidation markers in Trsp-deficient epithelial cells. This novel model of prostate neoplasia provides evidence for the existence of a selenoprotein or selenoproteins capable of acting as a tumor suppressor in the murine prostate.
Project description:Ferroptosis is form of regulated nonapoptotic cell death that is involved in diverse disease contexts. Small molecules that inhibit glutathione peroxidase 4 (GPX4), a phospholipid peroxidase, cause lethal accumulation of lipid peroxides and induce ferroptotic cell death. Although ferroptosis has been suggested to involve accumulation of reactive oxygen species (ROS) in lipid environments, the mediators and substrates of ROS generation and the pharmacological mechanism of GPX4 inhibition that generates ROS in lipid environments are unknown. We report here the mechanism of lipid peroxidation during ferroptosis, which involves phosphorylase kinase G2 (PHKG2) regulation of iron availability to lipoxygenase enzymes, which in turn drive ferroptosis through peroxidation of polyunsaturated fatty acids (PUFAs) at the bis-allylic position; indeed, pretreating cells with PUFAs containing the heavy hydrogen isotope deuterium at the site of peroxidation (D-PUFA) prevented PUFA oxidation and blocked ferroptosis. We further found that ferroptosis inducers inhibit GPX4 by covalently targeting the active site selenocysteine, leading to accumulation of PUFA hydroperoxides. In summary, we found that PUFA oxidation by lipoxygenases via a PHKG2-dependent iron pool is necessary for ferroptosis and that the covalent inhibition of the catalytic selenocysteine in Gpx4 prevents elimination of PUFA hydroperoxides; these findings suggest new strategies for controlling ferroptosis in diverse contexts.
Project description:Necrotic cell death during Mycobacterium tuberculosis (Mtb) infection is considered host detrimental since it facilitates mycobacterial spread. Ferroptosis is a type of regulated necrosis induced by accumulation of free iron and toxic lipid peroxides. We observed that Mtb-induced macrophage necrosis is associated with reduced levels of glutathione and glutathione peroxidase-4 (Gpx4), along with increased free iron, mitochondrial superoxide, and lipid peroxidation, all of which are important hallmarks of ferroptosis. Moreover, necrotic cell death in Mtb-infected macrophage cultures was suppressed by ferrostatin-1 (Fer-1), a well-characterized ferroptosis inhibitor, as well as by iron chelation. Additional experiments in vivo revealed that pulmonary necrosis in acutely infected mice is associated with reduced Gpx4 expression as well as increased lipid peroxidation and is likewise suppressed by Fer-1 treatment. Importantly, Fer-1-treated infected animals also exhibited marked reductions in bacterial load. Together, these findings implicate ferroptosis as a major mechanism of necrosis in Mtb infection and as a target for host-directed therapy of tuberculosis.
Project description:Phospholipid hydroperoxide glutathione peroxidase (GPx4) is a moonlighting selenoprotein, which has been implicated in basic cell functions such as anti-oxidative defense, apoptosis, and gene expression regulation. GPx4-null mice die in utero at midgestation, and developmental retardation of the brain appears to play a major role. We investigated post-transcriptional mechanisms of GPx4 expression regulation and found that the guanine-rich sequence-binding factor 1 (Grsf1) up-regulates GPx4 expression. Grsf1 binds to a defined target sequence in the 5'-untranslated region (UTR) of the mitochondrial GPx4 (m-GPx4) mRNA, up-regulates UTR-dependent reporter gene expression, recruits m-GPx4 mRNA to translationally active polysome fractions, and coimmunoprecipitates with GPx4 mRNA. During embryonic brain development, Grsf1 and m-GPx4 are coexpressed, and functional knockdown (siRNA) of Grsf1 prevents embryonic GPx4 expression. When compared with mock controls, Grsf1 knockdown embryos showed significant signs of developmental retardations that are paralleled by apoptotic alterations (TUNEL staining) and massive lipid peroxidation (isoprostane formation). Overexpression of m-GPx4 prevented the apoptotic alterations in Grsf1-deficient embryos and rescued them from developmental retardation. These data indicate that Grsf1 up-regulates translation of GPx4 mRNA and implicate the two proteins in embryonic brain development.
Project description:Selenium is an essential micronutrient in the diet of humans and other mammals. Based largely on animal studies and epidemiological evidence, selenium is purported to be a promising cancer chemopreventive agent. However, the biological mechanisms by which chemopreventive activity takes place are poorly understood. It remains unclear whether selenium acts in its elemental form, through incorporation into organic compounds, through selenoproteins or any combination of these. The purpose of this study was to determine whether selenoproteins mitigate the risk of developing chemically induced mammary cancer. Selenoprotein expression was ablated in mouse mammary epithelial cells through genetic deletion of the selenocysteine (Sec) tRNA gene (Trsp), whose product, designated selenocysteine tRNA, is required for selenoprotein translation. Trsp floxed and mouse mammary tumor virus (MMTV)-cre mice were crossed to achieve tissue-specific excision of Trsp in targeted mammary glands. Eight- to twelve-week-old second generation Trsp(fl/+);wt, Trsp(fl/+);MMTV-cre, Trsp(fl/fl);wt and Trsp(fl/fl);MMTV-cre female mice were administered standard doses of the carcinogen, 7,12-dimethylbenzylbenz[a]antracene. Our results revealed that heterozygous, Trsp(fl/+);MMTV-cre mice showed no difference in tumor incidence, tumor rate and survival compared with the Trsp(fl/+);wt mice. However, 54.8% of homozygous Trsp(fl/f)(l);MMTV-cre mice developed mammary tumors and exhibited significantly shorter survival than the corresponding Trsp(fl/fl);wt mice, where only 36.4% developed tumors. Loss of the homozygous Trsp alleles was associated with the reduction of selenoprotein expression. The results suggest that mice with reduced selenoprotein expression have increased susceptibility to developing carcinogen-induced mammary tumors and that a major protective mechanism against carcinogen-induced mammary cancer requires the expression of these selenoproteins.
Project description:The facets of host control during Plasmodium liver infection remain largely unknown. We find that the SLC7a11-GPX4 pathway, which has been associated with the production of reactive oxygen species, lipid peroxidation, and a form of cell death called ferroptosis, plays a critical role in control of Plasmodium liver stage infection. Specifically, blocking GPX4 or SLC7a11 dramatically reduces Plasmodium liver stage parasite infection. In contrast, blocking negative regulators of this pathway, NOX1 and TFR1, leads to an increase in liver stage infection. We have shown previously that increased levels of P53 reduces Plasmodium LS burden in an apoptosis-independent manner. Here, we demonstrate that increased P53 is unable to control parasite burden during NOX1 or TFR1 knockdown, or in the presence of ROS scavenging or when lipid peroxidation is blocked. Additionally, SLC7a11 inhibitors Erastin and Sorafenib reduce infection. Thus, blocking the host SLC7a11-GPX4 pathway serves to selectively elevate lipid peroxides in infected cells, which localize within the parasite and lead to the elimination of liver stage parasites.