Previously, we have demonstrated that the chloride channel ClC-2 modulates intestinal mucosal barrier function. In the present study, we investigated the role of ClC-2 in epithelial barrier development and maintenance in Caco-2 cells. During early monolayer formation, silencing of ClC-2 with small interfering (si)RNA led to a significant delay in the development of transepithelial resistance (TER) and disruption of occludin localization. Proteomic analysis employing liquid chromatography- mass spectrometry /mass spectrometry revealed association of ClC-2 with key proteins involved in intracellular trafficking, including caveolin-1 and Rab5. In ClC-2 siRNA-treated cells, occludin colocalization with caveolin-1 was diffuse and in the subapical region. Subapically distributed occludin in ClC-2 siRNA-treated cells showed marked colocalization with Rab5. To study the link between ClC-2 and trafficking of occludin in confluent epithelial monolayers, a Caco-2 cell clone expressing ClC-2 short hairpin (sh)RNA was established. Disruption of caveolae with methyl-β-cyclodextrin (MβCD) caused a marked drop in TER and profound redistribution of caveolin-1-occludin coimmunofluorescence in ClC-2 shRNA cells. In ClC-2 shRNA cells, focal aggregations of Rab5-occludin coimmunofluorescence were present within the cytoplasm. Wortmannin caused an acute fall in TER in ClC-2 shRNA cells and subapical, diffuse redistribution of Rab5-occludin coimmunofluorescence in ClC-2 shRNA cells. An endocytosis and recycling assay for occludin revealed higher basal rate of endocytosis of occludin in ClC-2 shRNA cells. Wortmannin significantly reduced the rate of recycling of occludin in ClC-2 shRNA cells. These data clearly indicate that ClC-2 plays an important role in the modulation of tight junctions by influencing caveolar trafficking of the tight junction protein occludin.
All Science Journal Classification (ASJC) codes
- Cell Biology