Abstract
Mucosal biopsies from inflamed colon of inflammatory bowel disease (IBD) patients exhibit elevated epithelial apoptosis compared to those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate IBD. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine: 1) The expression of PAR2 in IBD biopsies; 2) The role of PAR2 in preventing apoptosis of epithelial cell during colitis in vivo; and 3) whether PAR2 activation could rescue colonic epithelial cells from apoptosis induced by pro-apoptotic cytokines increased during IBD in vitro. PAR2 mRNA expression was decreased in colonic biopsies from inflamed IBD patients compared to healthy controls. Acute chemically-induced colitis stimulated greater colonic injury and inflammation in PAR2-null mice compared to wild-types. These effects were independent of intestinal microbial diversity and abundance. PAR2-null mice were also found to have increased numbers of apoptotic epithelial cells during colitis. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspases-3, 8, 9, poly-ADP ribose polymerase and the externalization of phosphatidylserine after treatment of HT-29 cells, derived from a human colonic adenocarcinoma, with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. PAR2 activation was found to increase phosphorylation and inactivation of pro-apoptotic BCL-2 antagonist of cell death (BAD) at Ser112 and Ser136 by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic myeloid cell leukemia 1 (MCL-1). Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, while single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing in IBD.
