title: Proteinase-activated receptor 2 (PAR2) decreases apoptosis in colonic epithelial cells.
abstract: Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, 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 proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, 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 Ser(112) and Ser(136) 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, whereas 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.
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Vadim
authors.initials: V
authors.lastname: Iablokov
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Christina L
authors.initials: CL
authors.lastname: Hirota
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Peplowski
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Rithwik
authors.initials: R
authors.lastname: Ramachandran
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Mihara
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada.
authors.forename: Morley D
authors.initials: MD
authors.lastname: Hollenberg
authors.affiliation: From the Department of Physiology and Pharmacology, the Inflammation Research Network, and the Snyder Institute for Chronic Diseases, University of Calgary, Alberta T2N 4N1, Canada wmacnaug@ucalgary.ca.
authors.forename: Wallace K
authors.initials: WK
authors.lastname: MacNaughton
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics*; D020013:Calcium Signaling; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003106:Colon / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005786:Gene Expression Regulation; D006706:Homeostasis / Q000187:drug effects; D006801:Humans; D007371:Interferon-gamma / Q000494:pharmacology; D048369:MAP Kinase Kinase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D048370:MAP Kinase Kinase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism; D010718:Phosphatidylserines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D044464:Receptor, PAR-2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D020169:Caspases; D007371:Interferon-gamma; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D009842:Oligopeptides; D019869:Phosphatidylinositol 3-Kinases; D010718:Phosphatidylserines; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011065:Poly(ADP-ribose) Polymerases; D034741:RNA, Small Interfering; D044464:Receptor, PAR-2; D014357:Trypsin; D014409:Tumor Necrosis Factor-alpha; C487404:2-furoyl-LIGRLO-amide; C109968:seryl-leucyl-isoleucyl--glycyl-lysyl-valine; C482128:MAP2K2 protein, human; C482125:MAP2K1 protein, human
keywords: Apoptosis; G Protein-coupled Receptor (GPCR); Intestinal Epithelium; Mitogen-activated Protein Kinase (MAPK); Protease