Proteinase-Activated Receptor 2 Decreases Apoptosis in Colonic Epithelial Cells [Molecular Bases of Disease]

October 20th, 2014 by Iablokov, V., Hirota, C. L., Peplowski, M. A., Ramachandran, R., Mihara, K., Hollenberg, M. D., MacNaughton, W. K.

Mucosal biopsies from inflamed colon of inflammatory bowel disease 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, but treatments that specifically target apoptotic pathways are lacking. 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 whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by pro-apoptotic cytokines that are increased during IBD. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspases-3, 8, 9, PARP and the externalization of phosphatidylserine after treatment of cells 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 both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser112 and Ser136 by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic 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.