Abstract
In obesity and diabetes, an imbalance in fatty acid uptake and fatty acid utilization leads to excess accumulation of lipid in non-adipose tissues. This lipid overload is associated with cellular dysfunction and cell death, which contribute to organ failure, a phenomenon termed lipotoxicity. To elucidate the molecular mechanism of lipid-mediated cell death, we generated and characterized a mutant Chinese hamster ovary cell line that is resistant to palmitate-induced cell death. In this mutant, random insertion of a retroviral promoter trap has disrupted the gene for the non-coding RNA, growth arrested DNA-damage inducible gene 7 (gadd7). Here we report that gadd7 is induced by lipotoxic stress in a reactive oxygen species (ROS)-dependent fashion and is necessary for both lipid- and general oxidative stress-mediated cell death. Depletion of gadd7 by mutagenesis or short hairpin RNA knockdown significantly reduces lipid and non-lipid induced ROS. Furthermore, depletion of gadd7 delays and diminishes ROS-induced endoplasmic reticulum stress. Together these data are the first to implicate a non-coding RNA in a feed-forward loop with oxidative stress and its induction of the endoplasmic reticulum stress response.
Highlights
Cellular homeostasis can be perturbed by a myriad of stimuli, including metabolic imbalance, oxidative stress, and aberrant protein folding
In an effort to elucidate how cells respond to lipid metabolic stress, we used retroviral promoter trap mutagenesis and selection in palmitate-supplemented media to isolate Chinese hamster ovary (CHO) cells that are resistant to lipotoxicity [21]
Disruption of gadd7 Confers Resistance to Lipotoxicity—We carried out a genetic screen in CHO cells using the ROSAgeo retroviral promoter trap vector to identify genes critical for the lipotoxic response by selecting for mutants in media supplemented with a pathophysiological concentration of palmitate (500 M palmitic acid complexed to bovine serum albumin at a 2:1 molar ratio) [21]
Summary
Cellular homeostasis can be perturbed by a myriad of stimuli, including metabolic imbalance, oxidative stress, and aberrant protein folding. We report that gadd7 is induced by lipotoxic stress in a reactive oxygen species (ROS)-dependent fashion and is necessary for both lipid- and general oxidative stressmediated cell death. Been missed in SDS-PAGE analyses, we generated gadd7 ORF At both 5 and 7 h of palmitate treatment, ROS levels were sigfusion constructs by replacing each predicted gadd7 stop codon nificantly increased, whereas gadd7 expression remained with a triple Myc epitope tag in-frame followed by a stop codon unchanged relative to untreated cells.
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