Epigenetic and Post-transcriptional Regulation of LYPD2 Modulates Smoking-associated Phenotype via CHRNA7 and DNMT1 in the Human Airway Epithelium

Background: The small airway epithelium (SAE, >6th generations), the initiation site of smoking-induced airway disorders, is highly sensitive to the stress of smoking. Lymphocyte antigen-6 (Ly6)/urokinase-type plasminogen activator receptor (uPAR, also called PLAUR) superfamily of proteins function in differentiation and inflammation, common phenotypes altered in smoking-related lung diseases including chronic obstructive pulmonary disease (COPD) and lung cancer. In the human small airway epithelial (SAE) transcriptome, LYPD2 (LY6/PLAUR domain containing 2), a gene with unknown function, is the most down-regulated Ly6/uPAR gene in smokers. Based on the knowledge that SLURP1 (secreted LY6/PLAUR domain containing 1), a paralog of LYPD2, modulates inflammatory phenotypes, we hypothesized that LYPD2 plays a role in the smoking-related phenotypes in the airway epithelium. RNA sequencing, single cell RNA sequencing and immunohistochemistry demonstrated that LYPD is expressed in airway epithelial cells, predominantly in club and intermediate cells. Quantification of LYPD2 expression in the airway epithelium demonstrated a significant suppression of LYPD2 in smokers, overall in the SAE, and specifically in club, mucous and intermedaite cells. Cigarette smoking extract (CSE) treatment of differentiating airway basal stem/progenitor cells (BC) in air liquid interface culture (ALI) demonstrated significantly down-regulated LYPD2 expression. LYPD2 expression correlated with the expression of tight junction and differentiation-related marker genes in vivo and LYPD2 down-regulation in airway epithelium in vitro resulted in an increased expression of smoking-related inflammatory genes accompanied by decreased airway transepithelial resistance, suppressed junctional gene expression, and diminished gene expression related to normal differentiation. We show the function of LYPD2 is likely mediated by CHRNA7 and DNMT1, associated with JNK and NF-κB signaling and can be modulated by miR-214-3p and epigenetic mechanisms.