Cell confluence-induced activation of signal transducer and activator of transcription-3 (Stat3) triggers epithelial dome formation via augmentation of sodium hydrogen exchanger-3 (NHE3) expression

Cell confluence induces the activation of signal transducer and activator of transcription-3 (Stat3) in various cancer and epithelial cells, yet the biological implications and the associated regulatory mechanisms remain unclear. Because confluent polarized epithelia demonstrate dome formation and sodium influx that mimic the onset of differentiation, we sought to elucidate the role of Stat3 in association with the regulation of selective epithelial transporters in this biological phenomenon. This study established the correlation between Stat3 activation and cell confluence-induced dome formation in Madin-Darby canine kidney cells (MDCK) by following Stat3 activation events in dome-forming cells. Epifluorescent and confocal microscopy provided evidence showing specific localization of phosphorylated Stat3 Tyr(705) in the nuclei of dome-forming cells at initial stages. The relationship was further elucidated by the establishment of tetracycline-inducible expression of constitutive Stat3 mutant (Stat3-C) in MDCK cells or expression of dominant negative Stat3 (Stat3-D) stable cell lines (MDCK and NMuMG). Dome formation was promoted by the expression of Stat3-C but inhibited by Stat3-D. Two trans-epithelial transporters, NHE3 and ENaC alpha-subunit, were found to be increased during cell confluence. Interestingly, NHE3 expression could be specifically up-regulated by Stat3-C but inhibited by Stat3-13 through promoter regulation, whereas NHE1 and ENaC alpha-subunit were not affected by Stat3 expression. Application of NHE3 shRNA, NHE3 inhibitors (EIPA and S3226) suppressed confluence-induced dome formation in MDCK or NMuMG cells. These results demonstrate a cell confluence-induced Stat3 signaling pathway in epithelial cells in triggering dome formation through NHE3 augmentation.