The Ability of Serine Proteases to Induce an Increase in Barrier Function is Dependent on the Tight Junction Protein Occludin

Abstract

Inflammatory mediators disrupt the tight junction and may contribute to the pathogenesis of IBD through modulation of barrier function. We have found that serine proteases such as trypsin and matriptase induce an increase in barrier function in intestinal epithelial cell lines. However we do not know if they can reverse barrier disruption induced by inflammation, or how they modulate components of the tight junction (TJ). The SCBN canine epithelial cell line was mounted in Ussing chambers and barrier function assessed by 4 kDa FITC‐dextran flux and transepithelial electrical resistance (TER) measurement. Apical treatment with trypsin (45 BAU/mL) or matriptase (0.5 BAU/mL) induces a rapid and sustained increase in TER and decrease in FITC‐dextran flux. Treatment of cells with IFNγ and TNFα for 48 hours induced a dose‐dependent increase in permeability and redistribution of claudin‐4, tricellulin, and occludin from the TJ, as assessed by confocal immunofluorescence. The higher the doses of cytokines, the less able trypsin or matriptase could induce an increase in TER or decrease in FITC flux. Knockdown of occludin using siRNA resulted in a 39.8% reduction in baseline TER but no change in FITC‐dextran flux. Occludin knockdown induced a 74.1% and 90.6% reduction in the ability of trypsin and matriptase, respectively, to induce an increase in TER. Phosphorylation status of myosin light chain was assessed by Phos‐tag acrylamide Western blotting and no changes were observed with serine protease treatment. Thus, during inflammatory conditions, cytokines alter tight junctional structure and occludin localization; causing serine proteases to lose their ability to enhance epithelial barrier function.