C2C12 transcriptome changes in response to hydrogen peroxide
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE3078 | GEO | 2005/08/11
SECONDARY ACCESSION(S): PRJNA93081
REPOSITORIES: GEO
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Project description:In muscle, reactive oxygen species (ROS) generation increases with strenuous activity, chronic unloading, and inflammatory stimuli; skeletal muscle function is very sensitive to ROS; and there are redox-sensitive signaling pathways. Using myogenic cell cultures, we asked whether hydrogen peroxide (H2O2) induces adaptive changes in skeletal muscle gene expression. H2O2 downregulated or failed to induce antioxidant or apoptotic genes in the myotubes. Instead, H2O2 changed the expression of genes for cytosolic and mitochondrial enzymes, and upregulated inflammatory mediators. Finally, H2O2 had a mostly inhibitory effect on the expression of many transcription factors. The results indicate that mild oxidative stress may induce an adaptive response in skeletal muscle without antioxidant upregulation or apoptosis. Experiment Overall Design: Cell Culture: Experiment Overall Design: C2C12 myoblasts (ATCC, Rockville, MD) were cultured in DMEM supplemented with 10% fetal calf serum at 37°C in 5% CO2. Myoblast differentiation was initiated by switching to differentiation medium (DMEM supplemented with 2% heat-inactivated horse serum) and allowed to continue for 96 hrs. Differentiated myotubes were treated for 2 hours with H2O2 (Sigma-Aldrich, St. Louis, MO) 0, 1, 10, 100, or 1000 uM in DMEM supplemented with 0.5% heat-inactivated horse serum. Experiment Overall Design: cDNA microarrays: Experiment Overall Design: Total RNA was obtained from treated C2C12 myotubes with TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's recommended protocol.Oligonucleotide microarray studies with Affymetrix Mouse Genome 430A gene chips (n=20 chips) were conducted as described earlier. Microarrays were washed and stained with a streptavidin-bound marker and scanned. Data were analyzed with Affymetrix Microarray Suite 5.0 software. Only genes with consistent absent/present calls in all four independent replicates per group were considered for further analysis. Treatment comparisons were crossed so that each H2O2 sample was compared to each control. The software uses the one-sided Wilcoxon's signed rank test to estimate increase/no change/ decrease difference calls for each pair-wise comparison. Only difference calls consistent in all pair-wise comparisons and with average changes > 1.75 were considered significant, resulting in a conservative list of genes with changed expression levels. Functional classification of genes was based on an extensive literature review.
2007-09-07 | E-GEOD-3078 | biostudies-arrayexpress
Project description:Our laboratory previously demonstrated that perivascular stem/stromal cells (CD146+ pericytes) can effectively recover muscle mass after a period of immobilization in young adult mice. However, cell-based therapies are problematic in aged mouse models due to lack of viability upon transplantation. Therefore, the purpose of this study was to develop a pericyte-based, cell-free strategy to recover muscle mass after disuse in aged mice. Single-cell RNA sequencing (scRNA-Seq) was performed on adult mouse skeletal muscle after two weeks of unilateral hindlimb immobilization, which revealed that muscle-resident pericytes uniquely upregulate the long noncoding RNA Malat1, a negative regulator of Nrf2, and fail to induce antioxidant gene expression in response to reactive oxygen species (ROS; H2O2). This information was used to guide the design of a strategy in which healthy donor pericytes were stimulated with ROS to produce small extracellular vesicles (EVs) that were subsequently transplanted into 4- and 24-26-month-old C57BL/6 mice after two weeks of unilateral hindlimb immobilization. H2O2-primed healthy muscle-derived pericytes produced EVs in culture that effectively reduced restored myofiber CSA in both adult (p=0.009) and aged (p=0.006) muscle after disuse. In contrast, unprimed pericyte-derived EVs did not influence myofiber size. Neither primed, nor unprimed EVs recovered capillary density, yet both stimulated collagen turnover. Healthy ROS-primed pericyte-derived small EVs effectively improve skeletal muscle recovery after immobilization, representing a novel cell-free approach to rebuild muscle mass in older adults after a period of disuse.
2022-09-28 | GSE185560 | GEO
Project description:Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. We have aimed at exploring the crosstalk between ROS and an EMT induced by TGFb in breast cancer cells. Methods: We observed that cells exposed to TGFb displayed a decrease of ROS levels. To identify the antioxidant pathways activated during EMT, we investigated the effect of long-term oxidative stress, for example by H2O2 (80 µM and 150 µM), on MTflECad epithelial cells derived from the MMTV-Neu mouse model. Results: We show that long-term treated MTflEcad epithelical cells with H2O2 display both higher migratory capability and tumorigenicity. Furthermore, we identified an activation of the Nrf2 transcription factor, master regulator of the antioxidant response, in H2O2-resistant cells, as well as in TGFb-induced mesenchymal cells. Conclusion: In this study we report the criticial role of the Nrf2-mediated glutathione metabolism in the protection of metastatic breast cancer cells from ferroptosis. Therapeutic targeting of antioxidant pathways may thus be beneficial for patients with metastatic disease.
2022-10-15 | GSE187476 | GEO
Project description:Background: Epithelial-mesenchymal transition (EMT) has been implicated in metastasis, drug resistance, survival under stress and also conferring stem cell-like traits to cancer cells. We have aimed at exploring the crosstalk between ROS and an EMT induced by TGFb in breast cancer cells. Methods: We observed that cells exposed to TGFb displayed a decrease of ROS levels. To identify the antioxidant pathways activated during EMT, we investigated the effect of long-term oxidative stress, for example by H2O2 (40 µM and 60 µM), on Py2T epithelial cells derived from the MMTV-PyMT mouse model. Results: We show that long-term treated Py2T epithelical cells with H2O2 display both higher migratory capability and tumorigenicity. Furthermore, we identified an activation of the Nrf2 transcription factor, master regulator of the antioxidant response, in H2O2-resistant cells, as well as in TGFb-induced mesenchymal cells. Conclusion: In this study we report the criticial role of the Nrf2-mediated glutathione metabolism in the protection of metastatic breast cancer cells from ferroptosis. Therapeutic targeting of antioxidant pathways may thus be beneficial for patients with metastatic disease.
2022-10-15 | GSE187364 | GEO
Project description:Toxoplasma gondii is an obligatory intracellular parasite that causes persistent infections in birds and mammals including ~30% of the world’s human population. Differentiation from proliferative and metabolically active tachyzoites to largely dormant bradyzoites initiates the chronic phase of infection and occurs predominantly in brain and muscle tissues. Here we used murine skeletal muscle cells (SkMCs) to decipher host cellular factors that favor T. gondii bradyzoite formation in terminally differentiated and syncytial myotubes, but not in proliferating myoblast precursors. Genome-wide transcriptome analyses of T. gondii-infected SkMCs and non-infected controls identified ~6,500 genes which were differentially expressed (DEGs) in myotubes compared to myoblasts, largely irrespective of infection. On the other hand, genes related to central carbohydrate metabolism, to redox homeostasis, and to the Nrf2-dependent stress response pathway were enriched in both infected myoblast precursors and myotubes. Stable isotope-resolved metabolite profiling indicated increased fluxes into the oxidative branch of the pentose phosphate pathway (OxPPP) in infected myoblasts and into the TCA cycle in infected myotubes. High OxPPP activity in infected myoblasts was associated with increased NADPH/NADP+ ratio while myotubes exhibited higher ROS levels and lower expression of anti-oxidants and detoxification enzymes. Pharmacological reduction of ROS levels in SkMCs inhibited bradyzoite differentiation, while increased ROS induced bradyzoite formation. Thus, we identified a novel host cell-dependent mechanism that triggers stage conversion of T. gondii into persistent tissue cysts in its natural host cell type.
2021-11-04 | GSE133952 | GEO
Project description:Ammonia is a cytotoxic metabolite with pleotropic molecular and metabolic effects in non-hepatic tissue/cells targeting mitochondrial oxidative function that may contribute to post-mitotic senescence. Global molecular responses were determined by analysis of unbiased data followed by experimental validation of critical functional consequences in hyperammonemic differentiated myotubes and skeletal muscle from the portacaval anastomosis (PCA) rat. Integrating whole cell transcriptome with whole cell and mitochondrial proteome from hyperammonemic myotubes identified oxidative dysfunction and senescence pathways as being the most enriched with differentially expressed genes/proteins. Hyperammonemia and ammonia-lowering result in distinct clusters of molecular responses. Functional and metabolic studies in hyperammonemic myotubes showed that defects in electron transport chain (ETC) complexes I and III, loss of supercomplex assembly, decreased ATP synthesis, increased free radical generation with oxidative modification of proteins/lipids and increased expression of senescence associated molecular phenotype (SAMP) that were partially reversed by ammonia lowering. Studies in muscle from PCA rats established physiological relevance of our cellular studies. Dysregulated ammonia metabolism causes mitochondrial dysfunction by transcriptional and translational perturbations in multiple pathways with a distinct skeletal muscle SAMP.
2021-11-22 | PXD027754 | Pride
Project description:Reactive oxygen species (ROS) are extensively assessed in physiological and pathological studies; however, the genes and mechanisms involved in antioxidant reactions are elusive. To address this knowledge gap, we used a forward genetic approach with mouse haploid embryonic stem cells (haESCs) to generate high-throughput mutant libraries, from which numerous oxidative stress-targeting genes were screened out. We performed proof-of-concept experiments to validate the potential inserted genes. Slc25a43 (one of the candidates) knockout (KO) ESCs presented reduced damage caused by ROS and higher cell viability when exposed to H2O2. Subsequently, ROS production and mitochondrial function analysis also confirmed that Slc25a43 was a main target gene of oxidative toxicity. In addition, we identified that KO of Slc25a43 activated mitochondria-related genes including Nlrx1 to protect ESCs from oxidative damage. Overall, our findings facilitated revealing target genes of oxidative stress and shed lights on the mechanism underlying oxidative death.
2022-03-16 | GSE198548 | GEO
Project description:Deep skeletal muscle proteome. Triceps muscle from C57BL6 mouse and C2C12 myotubes were measure by LC-MS.
2020-02-08 | MSV000084940 | MassIVE
Project description:The Saccharomyces cerevisiae Yap1p transcription factor is required for the H2O2-dependent activation of many antioxidant genes including the TRX2 gene encoding thioredoxin 2. To identify factors that regulate Yap1p activity, we carried out a genetic screen for mutants that show elevated expression of a TRX2-HIS3 fusion in the absence of H2O2. Two independent mutants isolated in this screen carried mutations in the TRR1 gene encoding thioredoxin reductase. Northern blot and whole-genome expression analysis revealed that the basal expression of most Yap1p targets and many other H2O2-inducible genes is elevated in Deltatrr1 mutants in the absence of external stress. In Deltatrr1 mutants treated with H2O2, the Yap1p targets, as well as genes comprising a general environmental stress response and genes encoding protein-folding chaperones, are hyperinduced. However, despite the elevated expression of genes encoding antioxidant enzymes, Deltatrr1 mutants are extremely sensitive to H2O2. The results suggest that cells lacking thioredoxin reductase have diminished capacity to detoxify oxidants and/or to repair oxidative stress-induced damage and that the thioredoxin system is involved in the redox regulation of Yap1p transcriptional activity.
2005-06-30 | E-SMDB-2716 | biostudies-arrayexpress
Project description:Sedanolide is a bioactive compound with anti-inflammatory and antitumor activities. Although it has been recently suggested that sedanolide activates the nuclear factor E2-related factor 2 (NRF2) pathway, there is little research on its effects on cellular resistance to oxidative stress. The objective of the present study was to investigate the function of sedanolide in suppressing hydrogen peroxide (H2O2)-induced oxidative damage and the underlying molecular mechanisms in human hepatoblastoma cell line HepG2 cells. We found that sedanolide activated the antioxidant response element (ARE)-dependent transcription mediated by the nuclear translocation of NRF2. Pathway enrichment analysis of RNA sequencing data revealed that sedanolide upregulated the transcription of antioxidant enzymes involved in the NRF2 pathway and glutathione metabolism. Then, we further investigated whether sedanolide exerts cytoprotective effects against H2O2-induced cell death. We showed that sedanolide significantly attenuated cytosolic and mitochondrial reactive oxygen species (ROS) generation induced by exposure to H2O2. Furthermore, we demonstrated that pretreatment with sedanolide conferred a significant cytoprotective effect against H2O2-induced cell death by preventing the decrease of the mitochondrial membrane potential and the increase of caspase-3/7 activity. Our study demonstrated that sedanolide enhanced cellular resistance to oxidative damage via the activation of the Keich-like ECH-associated protein 1 (KEAP1)–NRF2 pathway.
2023-11-29 | GSE245428 | GEO