Title: Autophagy mitigates ethanol-induced mitochondrial dysfunction and oxidative stress in esophageal keratinocytes.
Authors: Chandramouleeswaran, Prasanna M; Guha, Manti; Shimonosono, Masataka; Whelan, Kelly A; Maekawa, Hisatsugu; Sachdeva, Uma M; Ruthel, Gordon; Mukherjee, Sarmistha; Engel, Noah; Gonzalez, Michael V; Garifallou, James; Ohashi, Shinya; Klein-Szanto, Andres J; Mesaros, Clementina A; Blair, Ian A; Pellegrino da Silva, Renata; Hakonarson, Hakon; Noguchi, Eishi; Baur, Joseph A; Nakagawa, Hiroshi
Published In PLoS One, (2020)
Abstract: 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.
PubMed ID: 32966340
MeSH Terms: Animals; Autophagy*; Cell Line; Cells, Cultured; Esophagus/cytology*; Ethanol/pharmacology; Female; Keratinocytes/drug effects; Keratinocytes/metabolism*; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred C57BL; Microtubule-Associated Proteins/genetics; Microtubule-Associated Proteins/metabolism; Mitochondria/metabolism*; Oxidative Stress*; Protein Kinases/genetics; Protein Kinases/metabolism; Repressor Proteins/genetics; Repressor Proteins/metabolism; Signal Transduction; TOR Serine-Threonine Kinases/genetics; TOR Serine-Threonine Kinases/metabolism