Silencing of NHE2 Enhances Migratory Speed in Colonic Epithelial Cells

Abstract

BackgroundThe maintenance of epithelial function and barrier integrity is achieved by continuous renewal of the colonic epithelium through proliferation, migration and differentiation. Sodium hydrogen exchanger 2 (NHE2) is highly expressed in the colonic epithelium, where it is involved in Na+/H+ exchange, water absorption and pHi regulation. However, NHE2 deficiency in mice does not result in diarrheal phenotype. NHE2 is expressed in the cryptal region, where colonocytes exit the stem cell niche and migrate toward the surface.Aim and methodsTo study the role of NHE2 in colonocyte migration, we silenced NHE2 in the self‐differentiating Caco 2Bbe (C2Bbe) colonic cell line and studied the migration of cells using wound scratch assay. To analyze colonocyte migration during self‐renewal, NHE2−/− and WT mice were pulse‐labeled with bromodeoxyuridine (BrdU) and sacrificed at different time points. Sections taken from identical colonic segments were studied immunohistochemically.ResultsThe rate of colonocyte migration, defined by the occurrence of BrdU‐positive cells along the crypt‐villus axis was significantly higher in the colon of NHE2−/− mice compared to the control littermates. This was associated with a significant reduction in E‐cadherin and ZO‐1 expression in the basal parts of the crypts, pointing to alterations of the adhesion and tight junction formation of the colonocytes during the early stages of differentiation. Additionally, increased mRNA expression of β‐catenin was detected in isolated NHE2−/− colonic crypts compared to the control. To substantiate these findings, C2Bbe cells were stably transfected with shRNA, generating a cell line with ~70 % downregulated expression of NHE2. A significant increase of the migration rate, but reduced cell proliferation was observed in the NHE2 knock‐down compared to the mock transfected cells. This was accompanied with a significant decrease of E‐cadherin, but enhanced β‐catenin expression.ConclusionsOur results show that NHE2 is involved in the processes of colonocyte proliferation and migration along the crypt axis. Alterations in the cell adhesion formation, the Wnt/β‐catenin signaling pathway and disturbed formation of the E‐cadherin/β‐catenin complex, possibly induced by a lower pHi in NHE2‐deficient colonocytes, may be responsible for the increase in colonocyte migratory speed.Support or Funding InformationVolkswagen Foundation (VW‐Vorab), SFB621/C9, Se460/9‐4 and 21‐1.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Role of Plasma Membrane Sodium/Hydrogen Exchangers in Gastrointestinal Functions: Proliferation and Differentiation, Fluid/Electrolyte Transport and Barrier Integrity

“…The failed reestablishment of the tight junctions post injury, especially disrupted occludin and claudin-1 localization patterns in the nhe2 −/− tissue, was offered as a mechanistic explanation ( Moeser et al, 2008 ). Our group has used Caco2BBe cells to address the impact of NHE2 on epithelial restitution, and we found that Caco2BBe cells upon “wounding” of the monolayer exhibit “sheet” migration, which is significantly increased in Caco2BBe cells with downregulated NHE2 expression ( Nikolovska et al, 2018 ). In our model we have shown that the lack of NHE2 in Caco2BBe cells leads to a significant decrease of the intracellular pH i ( Yu et al, 2019 ; Zhou et al, 2021 ; Nikolovska et al, 2022 ), which might influence the generation of the tight junction network or different signaling pathways and therefore affect cell migration.…”