E-cigarette aerosols induce the hydrolysis of lysosomal glycerophospholipids through PLA2G4A activation initiated by nicotine binding to CHRNA3/α3 nAChR in airway epithelial cells

Accumulating evidence has demonstrated a significant association between e-cigarette exposure and airway epithelial damage. Nevertheless, the molecular drivers orchestrating this pathology remain unclear. Here, we demonstrated that nicotine is the key component of e-cigarette aerosols that induced pathogenic changes, including Apoptosis, oxidative stress, and mucus overproduction, in mouse airway epithelium and in human bronchial epithelial (HBE) cells. We further established that the nicotine of e-cigarette aerosols induced autophagosome formation via mTOR inhibition, while concurrently suppressing autolysosomal degradation through lysosomal membrane permeabilization (LMP). Restoration of lysosomal membrane integrity reversed e-cigarette aerosol-induced LMP and the subsequent macroautophagy/Autophagy inhibition, thereby alleviating airway epithelial damage. Mechanistically, nicotine of e-cigarette aerosols permeabilized lysosomal membranes via calcium-dependent activation of PLA2G4A, which hydrolyzed the sn-2 ester bond of lysosomal glycerophospholipids, generating lysophospholipids. This process was initiated by nicotine binding to CHRNA3/α3 nAChR, a ligand-gated ion channel whose activation triggered intracellular CA²⁺ overload. Genetic or pharmaceutical inhibition of CHRNA3 reduced intracellular CA²⁺ content, abolishing PLA2G4A activation. This inhibited lysosomal glycerophospholipid hydrolysis, thereby attenuating LMP and subsequently resolving autophagic flux blockade and cytotoxicity in HBE cells. Moreover, the role of CHRNA3-mediated PLA2G4A activation in e-cigarette aerosol-induced autophagy-lysosome dysfunction and cellular toxicity was validated in human lung organoids. Overall, our study underscores the importance of CHRNA3 activation, as a molecular initiating event (MIE), in the regulation of PLA2G4A-mediated hydrolysis of glycerophospholipids and autophagic flux impairment, and CHRNA3 inhibition could serve as a potential therapy for airway disorders induced by e-cigarette aerosols.Abbreviation: AACOCF3: arachidonyl trifluoromethyl ketone; AB-PAS: Alcian Blue Periodic Acid Schiff; ANXA5: annexin V; AOP: adverse outcome pathway; ATG: Autophagy related; BECN1: beclin 1; CASP3: Caspase 3; CASP7: Caspase 7; CASP9: Caspase 9; CQ: chloroquine; CHRNA/nAChR: cholinergic receptor nicotinic alpha subunit; CTSD: Cathepsin D; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; E-cigarette: electronic cigarette; ENGASE/NAG: endo-beta-N-acetylglucosaminidase; FBS: fetal bovine serum; GA: geldanamycin; GSH: glutathione; HBE: human bronchial epithelial; HEX: hexamethonium; LAMP: lysosome associated membrane protein; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LGALS3: Galectin 3; LBD: ligand-binding domain; LMP: lysosomal membrane permeabilization; LPC: lysophosphatidylcholine; LPE: lysophosphatidylethanolamine; MAP1LC3/LC3B: microtubule associated protein 1 light chain 3 beta; MDA: malondialdehyde; MIE: molecular initiating event; MTBE: methyl tert-butyl ether; MTOR: mechanistic target of rapamycin kinase; mtROS: mitochondrial reactive oxygen species; NBR1: NBR1 Autophagy cargo receptor; PBS: phosphate-buffered saline; PC: diacyl glycerophosphatidylcholine; PE: diacyl glycerophosphatidylethanolamine; Penh: pulmonary resistance; PG: propylene glycol; PLA2G4A/cPLA2: Phospholipase A2 group IVA; PLA2G4E: Phospholipase A2 group IVE; ROS: reactive oxygen species; siRNA: small interfering RNA; SOD: superoxide dismutase; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VG: vegetable glycerin.