Abstract 13684: The Function Of S100A8 In Human Primary Alveolar Epithelial Cells In Emphysema

Abstract

Pulmonary emphysema is characterized by the destruction of alveolar septa and impaired gas exchange. Cigarette smoking induces oxidative stress and is the main risk factor of lung injury and this disease development. There are limited therapeutic strategies for emphysema. Furthermore, cardiovascular diseases are the major cause of mortality in these patients. Alveolar type II (ATII) cells have stem cell potential to self-renew and regenerate the alveolar epithelium after damage. We isolated ATII cells from control nonsmoker and smoker organ donors and patients with emphysema to determine the nuclear role of S100A8. S100A8 is a small protein of 93 amino acids involved in cell growth and cytoprotection. We also used human alveolar epithelial A549 cells treated with cigarette smoke extract. Our results show increased oxidative stress as determined by 4-hydroxynonenal levels in A549 cells. We isolated cytoplasmic and nuclear fractions and observed that this exposure reduced S100A8 protein levels in the nucleus using Western blotting. This corresponded to our data using lung tissue and ATII cells obtained from smokers and emphysema patients. Moreover, we examined S100A8 nucleocytoplasmic shuttling and its stability using proteasome inhibitor and cycloheximide chase assay to determine protein function in the cytoplasm and nucleus in A549 cells. We immunoprecipitated S100A8 in A549 cells exposed to cigarette smoke and detected decreased S100A8 interaction with nuclear proteins by mass spectrometry analysis. We generated a stable A549 cell line with S100A8 knockout using the CRISPR/Cas9 strategy to determine its function. S100A8 ubiquitination and proteasomal degradation were also evaluated in human ATII cells isolated from control non-smokers, smokers, and emphysema. The findings improve our knowledge of the nuclear role of S100A8 in emphysema pathophysiology.