Objective
As a major cellular defense mechanism, the Nrf2/Keap1 pathway regulates expression of genes involved in detoxification and stress response. Our previous study revealed activation of the Nrf2/Keap1 pathway at the maturation phase during mouse esophageal development, suggesting a potential function in epithelial defense. Here we hypothesize that Nrf2 is involved in the barrier function of esophageal epithelium, and plays a protective role against gastroesophageal reflux disease (GERD).
Design
Human esophageal biopsy samples, mouse surgical models and Nrf2-/- mice were used to assess the role of the Nrf2/Keap1 pathway in esophageal mucosal barrier function. Trans-epithelial electrical resistance (TEER) was measured with mini-Ussing chambers. Hematoxylin and eosin (HE) staining and transmission electron microscopy were used to examine cell morphology, while gene microarray, immunohistochemistry, Western blotting and ChIP analysis were used to assess the expression of pathway genes.
Results
Nrf2 was expressed in normal esophageal epithelium and activated in GERD of both humans and mice. Nrf2 deficiency and gastroesophageal reflux in mice, either alone or in combination, reduced TEER and increased intercellular space diameter in esophageal epithelium. Nrf2 target genes and gene sets associated with oxidoreductase activity, mitochondrial biogenesis and energy production were down-regulated in the esophageal epithelium of Nrf2-/- mice. Consistent with the antioxidative function of Nrf2, a DNA oxidative damage marker (8OHdG) dramatically increased in esophageal epithelial cells of Nrf2-/- mice compared with those of wild-type mice. Interestingly, ATP biogenesis, Cox IV (a mitochondrial protein) and Claudin-4 (Cldn4) expression were down-regulated in the esophageal epithelium of Nrf2-/- mice, suggesting that energy-dependent tight junction integrity was subject to Nrf2 regulation. ChIP analysis confirmed the binding of Nrf2 to Cldn4 promoter.
Conclusion
Nrf2 deficiency impairs esophageal barrier function through disrupting energy-dependent tight junction. Elucidating the role of this pathway in GERD has potential implications for the pathogenesis and therapy of the disease.