Effects Of Nitric Oxide And Reactive Oxygen Species On Hif-1 Alpha Stabilization Following Clostridium Difficile Toxin Exposure Of The Caco-2 Epithelial Cell Line

Background/Aims: Stabilization of the hypoxia-inducible factor (HIF-1 alpha) 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-1 alpha stability and initiate protection against epithelial cell damage. Methods/Results: HIF-1 alpha 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-1 alpha protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W) attenuated HIF-1 alpha 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-1 alpha 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-1 alpha protein and nitrosylation levels. Conclusion: Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1 alpha and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury. Copyright (c) 2013 S. Karger AG, Basel