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Serine protease modulation of epithelial barrier function through the tight junction (650.8)
Author(s) -
Ronaghan Natalie,
MacNaughton Wallace
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.650.8
Subject(s) - occludin , trypsin , tight junction , ussing chamber , proteases , microbiology and biotechnology , chemistry , western blot , serine , serine protease , blot , protease , biology , phosphorylation , biochemistry , in vitro , enzyme , gene
Inflammatory mediator‐induced disruption of the tight junction may contribute to pathogenesis of IBD through increased intestinal permeability. We have previously found apical addition of the serine proteases trypsin and matriptase to colonic epithelial cell lines increase transepithelial electrical resistance (TER). We aimed to elucidate the role of tight junction (TJ) modulation in this phenomenon. Confluent monolayers of SCBN colonic epithelial cells were mounted on Ussing chambers after being transfected with siRNA against occludin and treated with 45 BAU/mL trypsin. Trypsin‐induced changes in TER and permeability to 4 kDa FITC‐dextran were monitored. siRNA reduced occludin levels by 90% (Western blot) and reduced the trypsin‐induced increase in TER to 29.8 ± 6.0% (n=4 p<0.05) of control response. However, no changes in trafficking of any TJ protein were observed by Triton X‐100 fractionation and Western blotting after trypsin treatment. In a calcium‐switch assay performed in Ussing chambers, trypsin did not induce faster recovery or have an affect on TER until 30 minutes post‐recovery of the monolayer. In conclusion, we have shown that occludin is essential in the serine protease‐mediated increase in TER but this is not through trafficking to the TJ, and that trypsin does not induce the formation of the tight junction. Grant Funding Source : Supported by Canadian Institutes of Health Research