Neutrophil elastase suppresses cigarette smoke extract‐induced activation of anti‐oxidative pathway by mediating nrf2 degradation in bronchial epithelial cells. (LB237)

Abstract

Cigarette smoke (CS) induces lung inflammation and simultaneously activates anti‐oxidative pathway as a defense mechanism. Recently, we found that neutrophil elastase (NE) enhances cigarette smoke extract (CSE)‐induced inflammation. Therefore, this study was undertaken to investigate the roles of NE on CSE‐induced activation of anti‐oxidative pathway and its regulatory mechanism in bronchial epithelial cells. CSE activated Nrf2, a transcription factor, which regulates the expression of several antioxidants. CSE increased anti‐oxidants such as HO‐1 and NQO1. Interestingly, NE suppressed CSE‐induced expression of HO‐1 mRNA and protein, but not NQO1. Therefore, we first determined the effects of NE on Nrf2. Although NE did not affect the basal and inducible level of Nrf2 mRNA, it significantly decreased the expression of Nrf2 protein. Nrf2 protein stability has been reported to be regulated by DJ‐1 and Keap1. However, the expression level of DJ‐1 and Keap1 did not change in NE‐treated cells. Nrf2 down‐regulation by NE was not blocked by treatment with proteasomal & lysosomal inhibitors (MG132, PS‐341 & chloroquine, NH4Cl), pan‐caspase inhibitor (zVAD‐fmk), or ROS blocker (NAC). Therefore, we hypothesized that down‐regulation of Nrf2 might be mediated by direct cleavage after NE internalization into cells. Immunofluorescence staining showed that NE was up‐taken intracellularly. Furthermore, in vitro fragmentation analysis supports that NE directly cleaved Nrf2. Taken together, these data suggest that NE accelerates CSE‐induced damage in lung epithelial cells by blocking anti‐oxidative pathway via Nrf2 proteolysis.