Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Abstract

Airway epithelial cells are exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen species (RNS) and reactive oxygen species (ROS). Previously we determined that the ROS, hydrogen peroxide (H2O2), activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in human airway epithelial (HAE) cells. Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. In the current study, we found that the RNS peroxynitrite (ONOO−) generated ceramide and induced apoptosis in HAE cells. Pretreatment with the antioxidant glutathione did not prevent ONOO−-induced apoptosis, but prevented H2O2-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. H2O2 specifically activated nSMase2 while ONOO−specifically activated acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H2O2-induced but not ONOO--induced apoptosis, whereas silencing of aSMase markedly impaired ONOO−-induced apoptosis, but did not affect H2O2-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different sphingomyelinases to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.