Nrf2 deficiency impairs the barrier function of mouse oesophageal epithelium

Abstract

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.

BMP-driven NRF2 activation in esophageal basal cell differentiation and eosinophilic esophagitis

“…Interestingly, loss of Keap1 leads to increased NRF2 activation and excessive differentiation of the epithelium in the embryonic esophagus and forestomach, where the lining epithelium is also stratified squamous (21)(22)(23)(24). In addition, a recent study showed that NRF2 activation is associated with the pathogenesis of GERD, and its deficiency impairs the maintenance of the barrier function of the epithelium (21), suggesting that the ROS pathway continues to play important roles in the adult esophagus. Here, we used 28,32).…”

“…We further showed that NRF2 is upregulated upon BMP activation and that inhibition of NRF2 activity attenuates BMP4-induced differentiation. Interestingly, constitutive activation of NRF2 in the esophagus of Keap1 -/-mutants also causes hyperkeratosis (21,22,24), which can be rescued by inhibition of…”

“…Upon oxidative stress, ubiquitination processes are blocked, leading to the stabilization and nuclear translocation of NRF2 and subsequent induction of NRF2 target genes including NAD(P)H quinine oxidoreductase-1 (Nqo1), which encodes an enzyme critical for reducing ROS levels (18)(19)(20). Interestingly, loss of Keap1 leads to increased NRF2 activation and excessive differentiation of the epithelium in the embryonic esophagus and forestomach, where the lining epithelium is also stratified squamous (21)(22)(23)(24). In addition, a recent study showed that NRF2 activation is associated with the pathogenesis of GERD, and its deficiency impairs the maintenance of the barrier function of the epithelium (21), suggesting that the ROS pathway continues to play important roles in the adult esophagus.…”

Gastrointestinal mucosal barrier function and diseases

“…A recent paper showed that mice deficient in Nrf2, a transcription factor related to oxidative stress, showed a loss of esophageal barrier function and a decrease in CLDN4 expression [30]. In that report, even though nuclear localization of Nrf2 in the granular layer in GERD was shown, the level of CLDN4 assayed was focused on the total amount in the mucosa, and CLDN4 staining was focused on the basal layers.…”

Surgical Models of Gastroesophageal Reflux with Mice

“…A low rate of postoperative survival does not allow experiments which require certain sample size to reach statistically sound conclusions. In the past we have successfully developed and characterized surgical models of gastric reflux, mixed reflux, duodenal reflux with mice in long-term experiments 9,11,12 . We have also provided consultation to several other groups in their mouse surgery.…”