Chymase mediated intestinal epithelial permeability is regulated by protease activating 1 receptor ( PAR ) - 2 / matrix metalloproteinase ( MMP ) - 2 - dependent mechanism

23 Mast cells regulate intestinal barrier function during disease and homeostasis. Homeostatic 24 regulation in vivo is through secretion of the mast cell-specific serine protease chymase. In the 25 present study, we employ in vitro model systems to delineate the molecular pathways involved in 26 chymase-mediated intestinal epithelial barrier dysfunction. Chymase stimulation of intestinal 27 epithelial cell monolayers (Caco2bbe) induced a significant reduction in transepithelial resistance 28 indicating decreased intestinal epithelial barrier function. The chymase-induced intestinal 29 epithelial barrier dysfunction was characterized by chymase-induced protease-activated receptor30 2 (PAR-2) activation and matrix metalloproteinase (MMP)-2 expression and activation. 31 Consistent with this observation, in vitro analysis revealed chymase induced PAR-2 activation, 32 and increased MAPK activity and MMP-2 expression. Pharmacological and siRNA-mediated 33 antagonism of PAR-2 and MMP-2 significantly attenuated chymase-stimulated barrier 34 dysfunction. Additionally, the chymase/MMP-2-mediated intestinal epithelial dysfunction was 35 associated with a significant reduction in the tight junction protein claudin-5, which was partially 36 restored by MMP-2 inhibition. Finally, incubation of Caco2bbe cells with chymase-sufficient, 37 but not with chymase-deficient bone marrow-derived mast cells (BMMCs) decreased barrier 38 function, which was attenuated by the chymase inhibitor, chymostatin. Collectively, these results 39 suggest that mast cell/chymase-mediated intestinal epithelial barrier function is mediated by 40 PAR-2/MMP-2-dependent pathways. 41 42