The serine protease-mediated increase in intestinal epithelial barrier function is dependent on occludin and requires an intact tight junction

Abstract

Barrier dysfunction is a characteristic of the inflammatory bowel diseases (IBD), Crohn's disease and ulcerative colitis. Understanding how the tight junction is modified to maintain barrier function may provide avenues for treatment of IBD. We have previously shown that the apical addition of serine proteases to intestinal epithelial cell lines causes a rapid and sustained increase in transepithelial electrical resistance (TER), but the mechanisms are unknown. We hypothesized that serine proteases increase barrier function through trafficking and insertion of tight junction proteins into the membrane, and this could enhance recovery of a disrupted monolayer after calcium switch or cytokine treatment. In the canine epithelial cell line, SCBN, we showed that matriptase, an endogenous serine protease, could potently increase TER. Using detergent solubility-based cell fractionation, we found that neither trypsin nor matriptase treatment changed levels of tight junction proteins at the membrane. In a fast calcium switch assay, serine proteases did not enhance the rate of recovery of the junction. In addition, serine proteases could not reverse barrier disruption induced by IFNγ and TNFα. We knocked down occludin in our cells using siRNA and found this prevented the serine protease-induced increase in TER. Using fluorescence recovery after photobleaching (FRAP), we found serine proteases induce a greater mobile fraction of occludin in the membrane. These data suggest that a functional tight junction is needed for serine proteases to have an effect on TER, and that occludin is a crucial tight junction protein in this mechanism.

Serine proteases at the cutting edge of IBD: Focus on gastrointestinal inflammation

“…While serine proteases have been suggested to mediate such effect through PKCζ activation, this level of regulation remains poorly understood. A key role in maintaining epithelial homeostasis was also ascribed to matriptase and prostasin, two serine proteases with trypsin‐like activity 41‐45 . In these studies, matriptase depletion, either by RNAi silencing in epithelial cells or genetic downregulation in St14 hypomorphic mice, compromises TJ and increases intestinal paracellular permeability 41,42 .…”

Occludin knockdown is not sufficient to induce transepithelial macromolecule passage

“…23 Based on studies in different epithelial cells, occludin seems to play a role in epithelial wound healing, 24 apoptosis 25 and to be important to the interaction with intraepithelial lymphocytes. 26 Studies addressing occludin's role in barrier function have suggested involvement in serine proteasedependent barrier modulation, 27 hypoxia-induced, 28 ethanol-induced 29 and hydrogen peroxideinduced reduction in barrier function. 30,31 Occludin and the epithelial leak pathway Interestingly, several studies observed no effect of occludin expression on basal flux of solutes across epithelia.…”

Cellular Tight Junctions Prevent Effective Campylobacter jejuni Invasion and Inflammatory Barrier Disruption Promoting Bacterial Invasion from Lateral Membrane in Polarized Intestinal Epithelial Cells

“…Earlier studies reported that Ca 2+ reintroduction recovered TER values and increased TJ integrity in EGTA-treated cells (Farshori and Kachar, 1999 ; Ronaghan et al, 2016 ). Therefore, we performed a calcium switch assay to examine the effect of TJ integrity on C. jejuni invasion.…”