Transcript Profiling Identifies Dynamic Gene Expression Patterns and an Important Role for Nrf2/Keap1 Pathway in the Developing Mouse Esophagus

Abstract

Background and AimsMorphological changes during human and mouse esophageal development have been well characterized. However, changes at the molecular level in the course of esophageal morphogenesis remain unclear. This study aims to globally profile critical genes and signaling pathways during the development of mouse esophagus. By using microarray analysis this study also aims to determine how the Nrf2/Keap1 pathway regulates the morphogenesis of the esophageal epithelium.MethodsGene expression microarrays were used to survey gene expression in the esophagus at three critical phases: specification, metaplasia and maturation. The esophagi were isolated from wild-type, Nrf2−/−, Keap1−/−, or Nrf2−/−Keap1−/− embryos or young adult mice. Array data were statistically analyzed for differentially expressed genes and pathways. Histochemical and immunohistochemical staining were used to verify potential involvement of the Wnt pathway, Pparβ/δ and the PI3K/Akt pathway in the development of esophageal epithelium.ResultsDynamic gene expression patterns accompanied the morphological changes of the developing esophagus at critical phases. Particularly, the Nrf2/Keap1 pathway had a baseline activity in the metaplasia phase and was further activated in the maturation phase. The Wnt pathway was active early and became inactive later in the metaplasia phase. In addition, Keap1−/− mice showed increased expression of Nrf2 downstream targets and genes involved in keratinization. Microarray and immunostaining data also suggested that esophageal hyperkeratosis in the Keap1−/− mice was due to activation of Pparβ/δ and the PI3K/Akt pathway.ConclusionsMorphological changes of the esophageal epithelium are associated with dynamic changes in gene expression. Nrf2/Keap1 pathway activity is required for maturation of mouse esophageal epithelium.

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

“…-/-mutants in which MAFs serve as mediators of NRF2 (22,52), further supporting the idea that functional activation of NRF2 is required for esophageal epithelial differentiation.…”

“…For X-gal staining, the esophagi were fixed in 4% paraformaldehyde for 30 minutes, followed by X-gal staining overnight at 37°C. The X-gal-stained samples were then dehydrated with ethanol and embedded in paraffin for sectioning as previously described (11,22). ISH analysis was performed as previously described (27,64).…”

“…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.…”

“…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…”

Gpr177 regulates pulmonary vasculature development

“…The secondary antibodies were either fluorescence-or DAB-conjugated, and images were acquired with a Leica SP1 confocal microscope. Wholemount X-gal staining was performed as previously described (Rodriguez et al, 2010;Chen et al, 2012).…”