Alkaline Ceramidase Mediates the Oxidative Stress Response in Drosophila melanogaster Through Sphingosine.
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ABSTRACT:
SUBMITTER: Zhang CH
PROVIDER: S-EPMC6529914 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Human cells respond to DNA damage by elevating sphingosine, a bioactive sphingolipid that induces programmed cell death (PCD) in response to various forms of stress, but its regulation and role in the DNA damage response remain obscure. Herein we demonstrate that DNA damage increases sphingosine levels in tumor cells by upregulating alkaline ceramidase 2 (ACER2) and that the upregulation of the ACER2/sphingosine pathway induces PCD in response to DNA damage by increasing the production of reactive oxygen species (ROS). Treatment with the DNA damaging agent doxorubicin increased both ACER2 expression and sphingosine levels in HCT116 cells in a dose-dependent manner. ACER2 overexpression increased sphingosine in HeLa cells whereas knocking down ACER2 inhibited the doxorubicin-induced increase in sphingosine in HCT116 cells, suggesting that DNA damage elevates sphingosine by upregulating ACER2. Knocking down ACER2 inhibited an increase in the apoptotic and necrotic cell population and the cleavage of poly ADP ribose polymerase (PARP) in HCT116 cells in response to doxorubicin as well as doxorubicin-induced release of lactate dehydrogenase (LDH) from these cells. Similar to treatment with doxorubicin, ACER2 overexpression induced an increase in the apoptotic and necrotic cell population and PARP cleavage in HeLa cells and LDH release from cells, suggesting that ACER2 upregulation mediates PCD in response to DNA damage through sphingosine. Mechanistic studies demonstrated that the upregulation of the ACER2/sphingosine pathway induces PCD by increasing ROS levels. Taken together, these results suggest that the ACER2/sphingosine pathway mediates PCD in response to DNA damage through ROS production.
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Project description:Broadly distributed species must cope with diverse and changing environmental conditions, including various forms of stress. Cosmopolitan populations of Drosophila melanogaster are more tolerant to oxidative stress than those from the species' ancestral range in sub-Saharan Africa, and the degree of tolerance is associated with an insertion/deletion polymorphism in the 3' untranslated region of the Metallothionein A (MtnA) gene that varies clinally in frequency. We examined oxidative stress tolerance and the transcriptional response to oxidative stress in cosmopolitan and sub-Saharan African populations of D. melanogaster, including paired samples with allelic differences at the MtnA locus. We found that the effect of the MtnA polymorphism on oxidative stress tolerance was dependent on the genomic background, with the deletion allele increasing tolerance only in a northern, temperate population. Genes that were differentially expressed under oxidative stress included MtnA and other metallothioneins, as well as those involved in glutathione metabolism and other genes known to be part of the oxidative stress response or the general stress response. A gene coexpression analysis revealed further genes and pathways that respond to oxidative stress including those involved in additional metabolic processes, autophagy, and apoptosis. There was a significant overlap among the genes induced by oxidative and cold stress, which suggests a shared response pathway to these two stresses. Interestingly, the MtnA deletion was associated with consistent changes in the expression of many genes across all genomic backgrounds, regardless of the expression level of the MtnA gene itself. We hypothesize that this is an indirect effect driven by the loss of microRNA binding sites within the MtnA 3' untranslated region.
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Project description:Overload of palmitic acids is linked to the dysregulation of ceramide metabolism in nonalcoholic steatohepatitis (NASH), and ceramides are important bioactive lipids mediating the lipotoxicity of palmitic acid in NASH. However, much remains unclear about the role of ceramidases that catalyze the hydrolysis of ceramides in NASH. By analyzing the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database, we found that alkaline ceramidase 3 (ACER3) is upregulated in livers of patients with NASH. Consistently, we found that Acer3 mRNA levels and its enzymatic activity were also upregulated in mouse livers with NASH induced by a palmitate-enriched Western diet (PEWD). Moreover, we demonstrated that palmitate treatment also elevated Acer3 mRNA levels and its enzymatic activity in mouse primary hepatocytes. In order to investigate the function of Acer3 in NASH, Acer3 null mice and their wild-type littermates were fed a PEWD to induce NASH. Knocking out Acer3 was found to augment PEWD-induced elevation of C18:1-ceramide and alleviate early inflammation and fibrosis but not steatosis in mouse livers with NASH. In addition, Acer3 deficiency attenuated hepatocyte apoptosis in livers with NASH. These protective effects of Acer3 deficiency were found to be associated with suppression of hepatocellular oxidative stress in NASH liver. In vitro studies further revealed that loss of ACER3/Acer3 increased C18:1-ceramide and inhibited apoptosis and oxidative stress in mouse primary hepatocytes and immortalized human hepatocytes induced by palmitic-acid treatment. These results suggest that ACER3 plays an important pathological role in NASH by mediating palmitic-acid-induced oxidative stress.
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Project description:p53 mediates the DNA damage response (DDR) by regulating the expression of genes implicated in cell cycle arrest, senescence, programmed cell death (PCD), and metabolism. Herein we demonstrate that human alkaline ceramidase 2 (ACER2) is a novel transcriptional target of p53 and that its transactivation by p53 mediates the DDR. We found that p53 overexpression or its activation by ionizing radiation (IR) upregulated ACER2 in cells. Two putative p53 responsive elements (p53REs) were found in its first intron of the ACER2 gene, and Chromatin Immunoprecipitation (ChIP) assays in combination with promoter activity assays demonstrated that these p53REs are the bona fide p53 binding sites that mediate ACER2 transactivation by p53. As ACER2 catalyzes the hydrolysis of ceramides into sphingosine, which in turn is phosphorylated to form sphingosine-1-phosphate (S1P), ACER2 upregulation increased the levels of both sphingosine and S1P while decreasing the levels of ceramides in cells. A moderate upregulation of ACER2 inhibited cell cycle arrest and cellular senescence in response to low-level expression of p53 or low-dose IR by elevating S1P, a pro-proliferative and pro-survival bioactive lipid, and/or decreasing ceramides whereas its robust upregulation mediated PCD in response to high-level expression of p53 or high-dose IR likely by accumulating cellular sphingosine, a pro-death bioactive lipid. ACER2 is frequently inactivated in various cancers due to its deletion or mutations, and restoring its expression inhibited the growth of tumor xenografts in mice. These results suggest that p53 mediates DDR and exerts its tumor suppressive role in part by regulating the expression of ACER2, which in turn regulates the bioactive sphingolipid lipids.
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Project description:Markers of oxidative damage have been detected in brain tissue from patients with Alzheimer disease (AD) and other neurodegenerative disorders. These findings implicate oxidative injury in the neurodegenerative process, but whether oxidative stress is a cause or a consequence of neurotoxicity remains unclear. We used a Drosophila model of human tauopathies to investigate the role of oxidative stress in neurodegeneration. Genetic and pharmacological manipulation of antioxidant defense mechanisms significantly modified neurodegeneration in our model, suggesting that oxidative stress plays a causal role in neurotoxicity. We demonstrate that the JNK signaling pathway is activated in our model, which is in agreement with previous findings in AD tissue. Furthermore, we show that the extent of JNK activation correlates with the degree of tau-induced neurodegeneration. Finally, our findings suggest that oxidative stress acts not to promote tau phosphorylation, but to enhance tau-induced cell cycle activation. In summary, our study identifies oxidative stress as a causal factor in tau-induced neurodegeneration in Drosophila.
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Project description:Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging.
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Project description:BackgroundAerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress.Methods and findingsWe used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genome-wide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67-79% and 56-66% of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis.ConclusionsWe identified novel candidate genes associated with variation in resistance to oxidative stress that have context-dependent effects. These results form the basis for future translational studies to identify oxidative stress susceptibility/resistance genes that are evolutionary conserved and might play a role in human disease.
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| S-EPMC6997559 | biostudies-literature
Project description:Broadly distributed species must cope with diverse and changing environmental conditions, including various forms of stress. Cosmopolitan populations of Drosophila melanogaster are more tolerant to oxidative stress than those from the species’ ancestral range in sub-Saharan Africa, and the degree of tolerance is associated with an insertion/deletion polymorphism in the 3’ untranslated region of the Metallothionein A (MtnA) gene that varies clinally in frequency. We examined oxidative stress tolerance and the transcriptional response to oxidative stress in cosmopolitan and sub-Saharan African populations of D. melanogaster, including paired samples with allelic differences at the MtnA locus. We found that the effect of the MtnA polymorphism on oxidative stress tolerance was dependent on the genomic background, with the deletion allele increasing tolerance only in a northern, temperate population. Genes that were differentially expressed under oxidative stress included MtnA and other metallothioneins, as well as those involved in glutathione metabolism and other genes known to be part of the oxidative stress response or the general stress response.
2022-01-11 | GSE174813 | GEO