title: 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.
authors: Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
authors: Natalie J
authors: NJ
authors: Ronaghan
authors: Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
authors: Judie
authors: J
authors: Shang
authors: Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
authors: Vadim
authors: V
authors: Iablokov
authors: Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
authors: Raza
authors: R
authors: Zaheer
authors: Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada
authors: Pina
authors: P
authors: Colarusso
authors: Département de Pharmacologie-Physiologie, Université de Sherbrooke, Sherbrooke, Quebec, Canada
authors: Sébastien
authors: S
authors: Dion
authors: Département de Pharmacologie-Physiologie, Université de Sherbrooke, Sherbrooke, Quebec, Canada
authors: Antoine
authors: A
authors: Désilets
authors: Département de Pharmacologie-Physiologie, Université de Sherbrooke, Sherbrooke, Quebec, Canada
authors: Richard
authors: R
authors: Leduc
authors: Departments of Pathology and Medicine (GI), Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts
authors: Jerrold R
authors: JR
authors: Turner
authors: Department of Physiology and Pharmacology and Calvin, Phoebe and Joan Snyder Institute for Chronic Diseases, Cumming School of Medicine, University of Calgary, Calgary, Alberta, Canada; and wmacnaug@ucalgary.ca.
authors: Wallace K
authors: WK
authors: MacNaughton
mesh_terms: D000818:Animals; D002460:Cell Line; D004285:Dogs; D017097:Electric Impedance; D055724:Electrophysiological Phenomena; D004847:Epithelial Cells / Q000166:cytology / Q000201:enzymology* / Q000502:physiology; D007413:Intestinal Mucosa / Q000166:cytology*; D062793:Occludin / Q000235:genetics / Q000378:metabolism*; D021381:Protein Transport; D012697:Serine Endopeptidases / Q000494:pharmacology; D057057:Serine Proteases; D062725:Tight Junction Proteins / Q000378:metabolism; D019108:Tight Junctions / Q000502:physiology*; D014357:Trypsin / Q000494:pharmacology
chemical_list: D062793:Occludin; D062725:Tight Junction Proteins; D057057:Serine Proteases; D012697:Serine Endopeptidases; C485595:matriptase; D014357:Trypsin
keywords: barrier function; inflammation; proteases; tight junction