3-Mercaptopyruvate Sulfurtransferase (MPST) Regulates Mitochondrial Metabolism and Epithelial Differentiation in Neonatal Patient-derived Airway Cells

Early-life airway epithelial development relies on tightly coordinated mitochondrial metabolic programs, yet the pathways that govern normal epithelial maturation during this vulnerable developmental window remain poorly defined. Defective epithelial maturation contributes to hyperoxia-induced lung injury, including the airway remodeling seen in infants with bronchopulmonary dysplasia (BPD), underscoring the need to identify mitochondrial pathways that regulate early epithelial differentiation. 3-Mercaptopyruvate sulfurtransferase (MPST), a mitochondrial sulfur metabolism enzyme, supports mitochondrial metabolic and bioenergetic function, but its role in neonatal airway epithelial development is unknown. Using neonatal patient-derived tracheal airway epithelial cells (nTAECs) in a 3D air-liquid interface (ALI) model, we first showed that hyperoxia reduces MPST and then dissected how MPST loss alters early epithelial differentiation metabolic homeostasis. MPST knockdown was induced by siRNA during ALI differentiation. Bulk RNA sequencing was performed on harvested cells, epithelial lineage markers were quantified by immunofluorescence, and metabolic flux was evaluated using Seahorse. MPST loss induced early (ALI day 3) transcriptomic shifts involving mitochondrial metabolic pathways, epithelial lineage programs, and stress-response signatures, with subsequently decreased ciliated cell lineages during mid-differentiation phase (ALI day 7). Metabolic flux analysis revealed significantly reduced mitochondrial respiration without compensatory increase in glycolysis, indicating disrupted metabolic flexibility. Together, these data show that MPST is essential for maintaining mitochondrial metabolic integrity necessary for normal airway epithelial development. Loss of MPST creates a developmental vulnerability that may contribute to hyperoxia-induced airway injury in neonates. Targeting MPST-dependent pathways could represent a new strategy to preserve airway health in infants at risk for BPD airway remodeling.