Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes
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ABSTRACT:
PROVIDER: PRJNA600739 | ENA |
REPOSITORIES: ENA
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Project description:The current study is focused on elucidating the effect of acute alcohol challenge on esophageal epithelial cells. We employed in vivo mouse models, ex-vivo 3D-organoids and 2D cell culture to answer this question. RNA-Seq was employed to identify acute alcohol-induced transcriptional changes in human esophageal epithelial cells in-vitro. Acute-alcohol promoted strong mitochondrial damage resulting in bioenergenitic imbalance in esophageal epithelial cells. Autophagy was activated potentially via the AMPK-mTORC1 pathway to promtoe the clearance of damaged mitochondria.
2020-10-01 | GSE143479 | GEO
Project description:During alcohol consumption, the esophageal mucosa is directly exposed to high concentrations of ethanol (EtOH). We therefore investigated the response of normal human esophageal epithelial cell lines EPC1, EPC2 and EPC3 to acute EtOH exposure. While these cells were able to tolerate 2% EtOH for 8 h in both three-dimensional organoids and monolayer culture conditions, RNA sequencing suggested that EtOH induced mitochondrial dysfunction. With EtOH treatment, EPC1 and EPC2 cells also demonstrated decreased mitochondrial ATPB protein expression by immunofluorescence and swollen mitochondria lacking intact cristae by transmission electron microscopy. Mitochondrial membrane potential (ΔΨm) was decreased in a subset of EPC1 and EPC2 cells stained with ΔΨm-sensitive dye MitoTracker Deep Red. In EPC2, EtOH decreased ATP level while impairing mitochondrial respiration and electron transportation chain functions, as determined by ATP fluorometric assay, respirometry, and liquid chromatography-mass spectrometry. Additionally, EPC2 cells demonstrated enhanced oxidative stress by flow cytometry for mitochondrial superoxide (MitoSOX), which was antagonized by the mitochondria-specific antioxidant MitoCP. Concurrently, EPC1 and EPC2 cells underwent autophagy following EtOH exposure, as evidenced by flow cytometry for Cyto-ID, which detects autophagic vesicles, and immunoblots demonstrating induction of the lipidated and cleaved form of LC3B and downregulation of SQSTM1/p62. In EPC1 and EPC2, pharmacological inhibition of autophagy flux by chloroquine increased mitochondrial oxidative stress while decreasing cell viability. In EPC2, autophagy induction was coupled with phosphorylation of AMP activated protein kinase (AMPK), a cellular energy sensor responding to low ATP levels, and dephosphorylation of downstream substrates of mechanistic Target of Rapamycin Complex (mTORC)-1 signaling. Pharmacological AMPK activation by AICAR decreased EtOH-induced reduction of ΔΨm and ATP in EPC2. Taken together, acute EtOH exposure leads to mitochondrial dysfunction and oxidative stress in esophageal keratinocytes, where the AMPK-mTORC1 axis may serve as a regulatory mechanism to activate autophagy to provide cytoprotection against EtOH-induced cell injury.
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Project description:Angiotensin IV mitigates diabetic cardiomyopathy via downregulating FoxO1-mediated autophagy
| PRJNA649543 | ENA
Project description:There is increasing evidence that autophagy contributes to the epidermal differentiation; however, the role of autophagy in epidermal tight junction (TJ) barrier remains unclear. To evaluate the role of autophagy in the maintenance of skin TJ barrier, we knocked out autophagy in human primary keratinocytes by infecting cells with autophagy-related gene 3 (Atg3) C264S mutant adenovirus.
2022-07-14 | GSE183921 | GEO
Project description:Autophagy is a mechanism that regulates cellular metabolism and clearance of damaged macromolecules and organelles. Impaired degradation of modified macromolecules contributes to cellular dysfunction and is observed in aged tissue and senescent cells. We have inactivated Atg7, an essential autophagy gene, in murine keratinocytes and have found in an earlier study that this resulted in increased baseline oxidative stress and reduced capacity to degrade crosslinked proteins after oxidative ultraviolet stress. To investigate whether autophagy deficiency would promote cellular aging, we studied, how Atg7 deficient (KO) and Atg7 bearing cells (WT) would respond to stress induced by Paraquat (PQ), an oxidant drug commonly used to induce cellular senescence.
2017-02-14 | GSE84935 | GEO
Project description:Activation of the systemic and myocardial rennin-angiotensin-aldosterone system (RAAS) by hyperglycemia plays a critical role in the development of diabetic cardiomyopathy. To test the hypothesis that AngIV protects against diabetic cardiomyopathy via stimulation of AT4R and inhibition of overactive autophagy, diabetic mice were treated with low-, medium- and high-dose AngIV, AT4R antagonist divalinal, forkhead box protein O1 (FoxO1) inhibitor AS1842856 (AS) or their combinations. In vitro, cardiomyocytes were treated with different concentrations of glucose, low-, medium- and high-dose AngIV, divalinal, FoxO1-overexpression plasmid (FoxO1-OE), AS or their combinations. The results showed that AngIV treatment dose-dependently attenuated left ventricular dysfunction, remodeling, fibrosis, and myocyte apoptosis in diabetic mice. Besides, autophagy and FoxO1 protein expression enhanced by diabetes were dose-dependently suppressed by AngIV treatment. However, these cardioprotective effects of AngIV were completely abolished by divalinal administration. Bioinformatic analyses revealed that the differentially expressed genes were enriched in autophagy, apoptosis, and FoxO signaling pathways among control, diabetes, and diabetes+high-dose AngIV groups. Similar to AngIV, AS treatment ameliorated diabetic cardiomyopathy in mice. In vitro, AngIV inhibited collagen expression, apoptosis, overactive autophagy flux, and FoxO1 nuclear translocation induced by high glucose in cardiomyocytes. However, these protective effects of AngIV were completely blocked by the use of divalinal or FoxO1-OE, and these detrimental effects were reversed by the additional administration of AS. In summary, AngIV treatment dose-dependently attenuated left ventricular dysfunction and remodeling in a mouse model of diabetic cardiomyopathy, and the mechanism involved stimulation of AT4R, suppression of FoxO1 nuclear translocation and inhibition of FoxO1-mediated overactive autophagy.
2020-07-30 | GSE155377 | GEO
Project description:Protein synthesis and autophagy are regulated by cellular ATP content. We tested the hypothesis that mitochondrial dysfunction, including generation of reactive oxygen species (ROS), contributes to impaired protein synthesis and increased proteolysis resulting in tissue atrophy in a comprehensive array of models. In myotubes treated with ethanol, using unbiased approaches, we identified defects in mitochondrial electron transport chain components, endogenous antioxidants, and enzymes regulating the tricarboxylic acid (TCA) cycle. Using high sensitivity respirometry, we observed impaired cellular respiration, decreased function of complexes I, II, and IV, and a reduction in oxidative phosphorylation in ethanol-treated myotubes and muscle from ethanol-fed mice. These perturbations resulted in lower skeletal muscle ATP content and redox ratio (NAD+/NADH). Ethanol also caused a leak of electrons, primarily from complex III, with generation of mitochondrial ROS and reverse electron transport. Oxidant stress with lipid peroxidation (thiobarbituric acid reactive substances) and protein oxidation (carbonylated proteins) were increased in myotubes and skeletal muscle from mice and humans with alcoholic liver disease. Ethanol also impaired succinate oxidation in the TCA cycle with decreased metabolic intermediates. MitoTEMPO, a mitochondrial specific antioxidant, reversed ethanol-induced mitochondrial perturbations (including reduced oxygen consumption, generation of ROS and oxidative stress), increased TCA cycle intermediates, and reversed impaired protein synthesis and the sarcopenic phenotype. We show that ethanol causes skeletal muscle mitochondrial dysfunction, decreased protein synthesis, and increased autophagy, and that these perturbations are reversed by targeting mitochondrial ROS.
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Project description:Expression data from autophagy-deficient keratinocytes and normal keratinocytes
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Project description:Study the influence of ethanol for human esophageal squamous epithelial cells.
2018-03-15 | GSE96734 | GEO
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