title: Effects of nitric oxide and reactive oxygen species on HIF-1α stabilization following clostridium difficile toxin exposure of the Caco-2 epithelial cell line.
abstract: OBJECTIVE
  Stabilization of the hypoxia-inducible factor (HIF-1α) is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C. difficile toxin exposure could influence HIF-1α stability and initiate protection against epithelial cell damage.
  
  
  RESULTS
  HIF-1α and inducible nitric oxide synthase (iNOS) proteins were up-regulated whereas factor-inhibiting HIF-1 (FIH-1) protein was down-regulated in Caco-2 epithelial cell monolayers with in vitro toxin exposure. We demonstrate using the biotin-switch assay that the stabilization of HIF-1α protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W) attenuated HIF-1α stabilization and exacerbated toxin-dependent disruptions in Caco-2 monolayer morphology and tight junctional integrity in vitro. Treatment of Caco-2 cell monolayers with N-actylcysteine (NAC), a scavenger of reactive oxygen species (ROS), attenuated toxin-dependent increases in iNOS and HIF-1α protein levels but had no effect on FIH-1 responses. In addition, mice that were exposed to C. difficile toxin in vivo also demonstrated a significant increase in HIF-1α protein and nitrosylation levels.
  
  
  CONCLUSIONS
  Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1α and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury.
authors.affiliation: Gastrointestinal Research Group at the Snyder Institute for Chronic Disease, Departments of Biochemistry & Molecular Biology, University of Calgary, Calgary, AB, Canada.
authors.forename: Joshua Y
authors.initials: JY
authors.lastname: Lee
authors.forename: Simon A
authors.initials: SA
authors.lastname: Hirota
authors.forename: Louise E
authors.initials: LE
authors.lastname: Glover
authors.forename: Glen D
authors.initials: GD
authors.lastname: Armstrong
authors.forename: Paul L
authors.initials: PL
authors.lastname: Beck
authors.forename: Justin A
authors.initials: JA
authors.lastname: MacDonald
mesh_terms: D000818:Animals; D001427:Bacterial Toxins / Q000633:toxicity*; D018938:Caco-2 Cells; D016360:Clostridioides difficile; D004847:Epithelial Cells / Q000187:drug effects*; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D015151:Immunoblotting; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D023421:Models, Animal; D009569:Nitric Oxide / Q000494:pharmacology*; D055550:Protein Stability / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000494:pharmacology*
chemical_list: D001427:Bacterial Toxins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D017382:Reactive Oxygen Species; D009569:Nitric Oxide