Abstract
Mucous cell metaplasia/hyperplasia in the middle ear epithelium is associated with the occurrence of otitis media with effusion during infections. However, the mechanism by which Notch signaling regulates cell fate in the middle ear epithelium is unclear. The aim of the present study was to elucidate this mechanism by investigating the localization of Notch receptors, such as Notch1 and Notch2, and Notch ligands, such as Jagged1, in the normal mouse middle ear epithelium (NMMEE) using immunofluorescence. Furthermore, the mRNA expression levels of Notch receptors and ligands were evaluated using reverse transcription polymerase chain reaction (PCR). The effects of the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine tert-butyl ester (DAPT) on epithelial cell proliferation were determined using 5-ethynyl-2'-deoxyuridine (EdU) staining and immunofluorescence staining of the apoptosis marker caspase-3 and the epithelial proliferation marker pan-cytokeratine. In addition, the differentiation of the NMMEE cells was characterized by evaluating the mRNA expression levels of the mucous cell-associated genes Arg2, Muc2, Spdef, Spink4 and Tff1 using quantitative PCR. Notch1, Notch2 and Jagged1 were observed to be co-localized throughout the mouse middle ear epithelium. Furthermore, Notch1-4, Jagged1, Jagged2, Dll1 and Dll4 mRNAs were expressed in the NMMEE cells. The inhibition of Notch by DAPT resulted in fewer EdU-positive cells and the upregulation of the expression levels of various mucous cell-associated genes. The results indicate that DAPT suppresses the proliferation of NMMEE cells while promoting their differentiation into mucous cells. Therefore, DAPT may provide a specific therapeutic strategy for the reversal of multiple pathological processes that are associated with epithelium thickening in the middle ear.