Bile salts disrupt human esophageal squamous epithelial barrier function by modulating tight junction proteins

Abstract

of acid and bile acids contributes to epithelial tissue injury in gastro-esophageal reflux disease. However, the influence of refluxed material on human esophageal stratified epithelial barrier function and tight junction (TJ) proteins has not been fully elucidated. Here, we investigated the influence of acid and bile acids on barrier function and TJ protein distribution using a newly developed air-liquid interface (ALI) in vitro culture model of stratified squamous epithelium based on primary human esophageal epithelial cells (HEECs). Under ALI conditions, HEECs formed distinct epithelial layers on Transwell inserts after 7 days of culture. The epithelial layers formed TJ, and the presence of claudin-1, claudin-4, and occludin were detected by immunofluorescent staining. The NP-40-insoluble fraction of these TJ proteins was significantly higher by day 7 of ALI culture. Exposure of HEECs to pH 2, and taurocholic acid (TCA) and glycocholic acid (GCA) at pH 3, but not pH 4, for 1 h decreased transepithelial electrical resistance (TEER) and increased paracellular permeability. Exposure of cell layers to GCA (pH 3) and TCA (pH 3) for 1 h also markedly reduced the insoluble fractions of claudin-1 and -4. We found that deoxycholic acid (pH 7.4 or 6, 1 h) and pepsin (pH 3, 24 h) significantly decreased TEER and increased permeability. Based on these findings, ALIcultured HEECs represent a new in vitro model of human esophageal stratified epithelium and are suitable for studying esophageal epithelial barrier functions. Using this model, we demonstrated that acid, bile acids, and pepsin disrupt squamous epithelial barrier function partly by modulating TJ proteins. These results provide new insights into understanding the role of TJ proteins in esophagitis. esophageal epithelium; claudin; air-liquid interface THE HUMAN ESOPHAGUS IS LINED with a layer of nonkeratinizing stratified squamous epithelium, which serves as an effective barrier against the influx of luminal contents. The esophageal epithelium consists of the basal, suprabasal, and superficial layers (14). In the pathogenesis of reflux esophagitis, acids, bile acids, and pepsin play a major role in damaging the esophageal epithelium (34). However, the mechanisms by which the luminal contents affect the barrier function of the stratified epithelial cell layers remain unclear.Tight junctions (TJs) separate the apical and basolateral cell surface domains to establish cell polarity and establish barrier function. TJs are composed of several proteins, including occludin, claudins, junctional adhesion molecule (22), and the scaffold protein zonula occludens (ZO). Of these proteins, claudins, which form specialized cellular structures that regulate the permeability of cellular layers, are the most important structural and functional components of TJ strands (1,6,10,30). Claudins are an integral transmembrane protein family consisting of at least 27 subtypes in mammals (22,40) and are essential for establishing and maintaining epithelial cell polarity by controlling the m...

Gastrointestinal mucosal barrier function and diseases

“…We also examined cell morphological changes at these time points. When granular layers started to develop, the TJ proteins occludin and ZO-1 began to be expressed in these layers as dots and whisker lines [14,15]. These data are consistent with the localization of these TJ proteins in granular layers in the human esophagus.…”

“…We further demonstrated that stratified layers representing basal and prickle cell layers began to develop after 3 days of culture, and staining showed that these layers were lined with CLDN1 and CLDN4. However, a barrier function was totally absent in the layers on these days, indicating that expression of these TJ proteins and their localization as a lining of the cell membrane of the layers do not have a barrier function [14,15]. The data also indicate that the DIS located at the basal and suprabasal layers does not contribute to the specific function of mucosal permeability (Fig.…”

“…3). CLDN1 and CLDN4 are stained as dense dots and whisker lines in granular layers, and show a barrier function when granular layers exist [13][14][15]17]. It is reasonable to assume that layers showing such dense staining of TJ proteins in these cultures have functioning TJs, in a manner similar to that in monolayer cells.…”

“…In evaluating the function of esophageal epithelial layers, we originally established esophageal stratified epithelial cell layers in vitro [12][13][14][15][16]. When we observed the development of stratified epithelial layers, we found that the barrier was dramatically developed 5 to 7 days after air-liquid interface (ALI)-culture of primary human squamous epithelial cells [15].…”

“…When we observed the development of stratified epithelial layers, we found that the barrier was dramatically developed 5 to 7 days after air-liquid interface (ALI)-culture of primary human squamous epithelial cells [15]. We also examined cell morphological changes at these time points.…”