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Autophagy involves the lysosomal degradation of cytoplasmic contents for regeneration of anabolic substrates during nutritional or inflammatory stress. Its initiation occurs rapidly after inactivation of the protein kinase mammalian target of rapamycin (mTOR) (or mechanistic target of rapamycin), leading to dephosphorylation of Unc-51-like kinase 1 (ULK1) and autophagosome formation. Recent studies indicate that mTOR can, in parallel, regulate the activity of stress transcription factors, including signal transducer and activator of transcription-1 (STAT1). The current study addresses the role of STAT1 as a transcriptional suppressor of autophagy genes and autophagic activity. We show that STAT1-deficient human fibrosarcoma cells exhibited enhanced autophagic flux as well as its induction by pharmacological inhibition of mTOR. Consistent with enhanced autophagy initiation, ULK1 mRNA and protein levels were increased in STAT1-deficient cells. By chromatin immunoprecipitation, STAT1 bound a putative regulatory sequence in the ULK1 5'-flanking region, the mutation of which increased ULK1 promoter activity, and rendered it unresponsive to mTOR inhibition. Consistent with an anti-apoptotic effect of autophagy, rapamycin-induced apoptosis and cytotoxicity were blocked in STAT1-deficient cells but restored in cells simultaneously exposed to the autophagy inhibitor ammonium chloride. In vivo, skeletal muscle ULK1 mRNA and protein levels as well as autophagic flux were significantly enhanced in STAT1-deficient mice. These results demonstrate a novel mechanism by which STAT1 negatively regulates ULK1 expression and autophagy., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: To evaluate the effects of physiological levels of mitochondrial-derived reactive oxygen species (ROS) on skeletal muscle autophagy, a proteolytic pathway designed to regulate contractile and myofilament homeostasis and to recycle long-lived proteins and damaged organelles., Results: Basal levels of autophagy and autophagy triggered by 1.5 to 4 h of acute nutrient deprivation, rapamycin treatment, or leucine deprivation were measured in differentiated C2C12 myotubes using long-lived protein degradation assays, LC3B lipidation, autophagy-related gene expression, and electron microscopy. Preincubation with the general antioxidants tempol (superoxide dismutase mimic) and N-acetyl cysteine (NAC) or the mitochondria-specific antioxidants mito-tempol and SS31 significantly decreased the rates of long-lived protein degradation and LC3B flux and blocked the induction of autophagy-related gene expression. Mitochondrial ROS levels significantly increased in response to acute nutrient deprivation and rapamycin treatment. Mito-tempol and tempol blocked this response. Antioxidants decreased AMP-activated protein kinase (AMPK) phosphorylation by 40% and significantly increased protein kinase B (AKT) phosphorylation, but exerted no effects on mTORC1-dependent ULK1 phosphorylation on Ser(555). NAC significantly decreased basal LC3B autophagic flux in skeletal muscles of mice., Innovation: We report for the first time that endogenous ROS promote skeletal muscle autophagy at the basal level and in response to acute nutrient starvation and mTORC1 inhibition. We also report for the first time that mitochondrial-derived ROS promote skeletal muscle autophagy and that this effect is mediated, in part, through regulation of autophagosome initiation and AKT inhibition., Conclusion: Mitochondrial-derived ROS promote skeletal muscle autophagy and this effect is mediated, in part, through activation of AMPK and inhibition of AKT.