Regulation of intestinal epithelial apoptosis by protease‐activated receptor 2 involves EGFR‐transactivation and is associated with Mcl‐1 upregualtion (151.1)

Abstract

Colonic biopsies from inflamed colon have higher levels of epithelial apoptosis than biopsies from healthy individuals. Therapies that inhibit intestinal epithelial apoptosis may decrease the inflammation observed in inflammatory bowel disease. We previously showed that signaling by PAR2, a GPCR activated by serine proteases, delayed IFN‐γ & TNF‐α‐induced apoptosis in colonic epithelial (HT‐29) cells. The observed decrease in the cleavage of caspase‐8, 9, 3 and PARP following the activation of PAR2 by 2‐furoyl‐LIGRLO (2fLI) was shown to be dependent on the activities of PI3K and ERK1/2. In this study we found that selective inhibition of EGFR with AG1478 blocked the phosphorylation of PI3K and ERK1/2. Pre‐treatment with AG1478 prior to the addition of IFN‐γ & TNF‐α also blocked 2fLI‐induced intestinal epithelial survival, as determined by PARP cleavage. Modulation of BCL‐2 family members was then investigated due to their known function in regulating mitochondrial permeability and apoptosis. Protein expression of pro‐apoptotic BAK, BAX and BIM were not altered by either cytokine or 2fLI treatment. Likewise, no change in expression of anti‐apoptotic BCL‐XL was observed. Treatment with 2fLI stimulated phosphorylation and inactivation of the apoptotic BH3‐only protein, BAD. Knockdown of BAD with siRNA, however, did not alter 2fLI‐stimulated intestinal survival. Finally, 2fLI stimulated a rapid increase in anti‐apoptotic MCL‐1 expression. PAR2 signaling reduces epithelial apoptosis in response to inflammatory cytokines via EGFR transactivation and possibly by regulation of MCL‐1 expression. Our findings may represent a mechanism whereby proteases facilitate epithelial cell survival and colonic healing after inflammation.Grant Funding Source: Supported by the Crohn’s and Colitis Foundation of Canada