NIX-associated mitochondrial remodeling contributes to esophageal epithelial differentiation

Abstract Mitochondria are increasingly recognized as regulators of cellular differentiation, but their role in esophageal epithelial homeostasis remains poorly understood. Here, we investigated mitochondrial remodeling during esophageal epithelial differentiation and whether mitochondrial depletion contributes to the acquisition of the differentiated phenotype. Mitochondrial abundance was assessed across normal human esophageal epithelium and in non-transformed immortalized human esophageal epithelial cells (EPC2-hTERT) using two differentiation models. Mitochondrial architecture was quantified, and the functional role of mitochondrial abundance was examined using doxycycline-inducible depletion of transcription factor A, mitochondrial (TFAM). Mitochondrial abundance progressively decreased from basal to superficial compartments of normal human esophageal epithelium and during in vitro differentiation, accompanied by fragmentation and remodeling of the mitochondrial network. TFAM depletion reduced mitochondrial abundance and increased squamous differentiation markers, indicating that mitochondrial depletion was sufficient to promote differentiation. Analysis of candidate mitochondrial clearance pathways identified Bcl2-interacting protein 3-like ( BNIP3L )/NIX (NIP3-like protein X) as preferentially associated with differentiated epithelial cells and induced during differentiation. NIX was associated with mitochondria and increased as mitochondrial abundance declined. NIX depletion prevented differentiation-associated mitochondrial depletion and attenuated differentiation marker induction. In human esophageal epithelium, NIX expression increased across the basal-to-suprabasal compartment before declining superficially. BNIP3L expression was reduced in active eosinophilic esophagitis (EoE) and increased following corticosteroid-associated remission. Interleukin-13 similarly reduced BNIP3L expression and suppressed epithelial differentiation in EPC2-hTERT cells. These findings identify NIX-associated mitochondrial remodeling as an important component of esophageal epithelial differentiation and suggest that disruption of mitochondrial quality control may contribute to impaired epithelial differentiation in EoE. New and Noteworthy Although impaired esophageal epithelial differentiation is associated with various esophageal diseases, mechanisms regulating differentiation under homeostasis remain incompletely understood. We show that mitochondria are progressively cleared during esophageal epithelial differentiation and that mitochondrial depletion is sufficient to promote differentiation. BNIP3L/NIX regulates mitochondrial clearance during this process. Moreover, BNIP3L expression is reduced in active eosinophilic esophagitis (EoE) and restored with remission, linking disrupted mitochondrial quality control to epithelial dysfunction in EoE.