ATP8B1 Deficiency Enhances Hyperoxia-Induced Lipid Oxidation and Apoptotic Response in Lung Epithelial Cells
Saturday, March 5, 2016
South Exhibit Hall H (Convention Center)
Andrew J. Cooke, MD, Jutaro Fukumoto, MD and PhD, Lee C Tan, Richard F. Lockey, MD FAAAAI, Narasaiah Kolliputi, PhD

Idiopathic pulmonary fibrosis (IPF) is a progressive, fibrotic lung disease with a need for novel models to study its pathogenesis.  Preliminary studies indicate that mice with loss-of-function mutation at the ATP8B1 gene develop  pulmonary fibrosis under hyperoxic conditions.  This study determined if hyperoxia (which causes oxidative stress) causes ATP8B1 knock-down (KD) LA4 cells to dedifferentiate into a profibrotic phenotype.  


ATP8B1-KD LA4 cells and empty plasmid-treated controls were exposed to normal versus hyperoxia for 48 hours.   The protein extracts from whole cell lysate were evaluated by western blot to assess the expression of proteins, including those related to apoptosis (BAD and Bcl-xL), epithelial cell characteristics (pro-SPC), oxidative stress (Nrf2 and 4-HNE), and mesenchymal expression (vimentin).


ATP8B1-KD LA4 cells showed an increased expression of the pro-apoptotic protein, BAD, and decreased expression of the anti-apoptotic protein, Bcl-xL, when compared to controls at 48 hrs of hyperoxia versus normoxia.   Similarly, these cells compared to control cells exhibited decreased epithelial properties with lower expression of pro-SPC.  Likewise, expression of Nrf2, a marker for response to oxidative stress was decreased, and 4-HNE, a marker for peroxidized lipid, was increased.   Finally, the expression of vimentin was increased when compared to controls. 


These results suggest that under oxidative conditions, ATP8B1 deficiency compromises the oxidative stress resistance mechanisms of these lung epithelial cells causing them to dedifferentiate into a profibrotic phenotype.