NOVEL REGULATION OF A PAR2‐MEDIATED CELLULAR MIGRATION PROGRAM BY PRO‐INFLAMMATORY CYTOKINES

Abstract

BACKGROUNDMucosal healing is the gold standard for IBD therapy. However, the mechanisms that determine mucosal healing are complex and need to be better understood to explain why many patients fail to achieve this key goal of therapy. The inflammatory environment is characterized by the presence of several serine proteases that signal through the protease‐activated receptors (PARs). PAR2 is ubiquitously expressed in the epithelial cells of the intestinal mucosa and activation serves a protective role consistent with host defence and repair. While our preliminary findings indicate that activation of PAR2 enhances wound closure, we do not know the cellular mechanisms that underlie this response.We hypothesize that activation of PAR2 induces a cellular migration program in intestinal epithelial cells that enhances mucosal healing in the inflammatory milieu.METHODSCircular wounds were introduced in T84 colonic epithelial cells. Wounded monolayers were treated with the PAR2 activating peptide, 2‐furoyl‐LIGRLO (2fLI, 5 μM), or the control reverse‐sequence peptide, 2‐furoyl‐OLRGIL (2fO, 5 μM), and live cell imaging was utilized to record wound closure over a 24 hr period. In other experiments, a cytokine cocktail consisting of IFNγ (10 ng/mL) and TNFα (10 ng/mL), alone or in combination with 2fLI, was applied to wounded monolayers and wound closure assessed. Intracellular effects of PAR2 activation and cytokine treatment were evaluated by measuring phosphorylation of key canonical signaling proteins using the MSD multi‐plex ELISA array system. EdU staining, with or without Mitomycin C (MMC), an inhibitor of proliferation, was used to determine the effect of cellular proliferation on wound closure.RESULTSPAR2 activation by 2fLI promoted wound closure in a concentration‐dependent manner (0.1 μM – 10 μM) compared to 2fO controls at the 12 and 24 hr timepoints. Interestingly, co‐stimulation with 2fLI and the cytokine cocktail resulted in an enhanced wound closure response at the 12 and 24 hr timepoints that was significantly greater than individual treatments (p<0.001). Co‐stimulation activated the downstream intracellular targets ERK1/2 and JNK compared to 2fO (p<0.001), but this activation was not greater than stimulation with 2fLI or cocktail alone, suggesting that MAP kinase activation was not responsible for the enhanced effect. MMC pre‐treatment partially reduced wound closure caused by co‐stimulation with 2fLI/IFNγ/TNFα, indicating that the enhanced wound healing effect was due in part to increased proliferation.CONCLUSIONSThese data suggest that both PAR2 activation and cytokine treatment promote wound closure in vitro through both enhanced cell migration and proliferation. The enhanced wound healing response to PAR2 activation in the presence of pro‐inflammatory cytokines suggests that the inflammatory milieu is necessary to initiate a proper wound healing response.Support or Funding InformationCanadian Institutes of Health Research (CIHR)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.