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Protease‐Activated Receptor 2 Drives Colonic Epithelial Wound Healing via EGFR Transactivation
Author(s) -
Bandara Mahesha N.S.,
MacNaughton Wallace Keith
Publication year - 2019
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2019.33.1_supplement.662.40
Subject(s) - wound healing , receptor , epidermal growth factor receptor , biology , proteases , protease activated receptor 2 , transactivation , epithelium , microbiology and biotechnology , immunology , gene expression , biochemistry , genetics , enzyme linked receptor , gene , enzyme
Background Compromised intestinal barrier function is a prominent feature of inflammatory bowel diseases. Epithelial restitution is required in the resolution of inflammation. For this to occur epithelial cells need to transition to migratory phenotype from its normal barrier phenotype. The inflammatory milieu of the intestinal epithelium contains a variety proteases which are upregulated in IBD patients and which are agonists of protease activated receptors (PARs). PAR2 is membrane‐spanning cell surface receptor which is widely present in the gastrointestinal tract. PAR2 activation can induce expression of cyclooxygenase‐2 (COX‐2) and transactivation of epidermal growth factor receptor (EGFR) in the intestinal epithelium. However, the specific roles of PAR2 activation in IBD remain unclear. Hypothesis We hypothesized that PAR2 activation drives COX‐2 and EGFR dependent epithelial wound healing. Methods CMT93 mouse intestinal epithelial cells were grown to confluence. PAR2 mRNA expression was detected by RT‐PCR and confirmed by cDNA sequencing. PAR2 A5 antibody was used to detect total PAR2 by immunocytochemistry and the tight junction protein, ZO‐1, demarcated apical and basolateral surfaces. PAR2 was activated by selective activating peptide 2‐furoyl‐LIGRLO‐NH2 (2fLI). Functionality of PAR2 was tested by measuring intracellular Ca 2+ , since it has been shown that PAR2 activation elicits Ca 2+ ‐dependent signaling. Scratch wounds were made in post‐confluent CMT93 monolayers and imaged using the IncuCyte™ live‐cell imaging system over 24 hours. COX‐2 protein was measured by western blotting and inhibited in wound healing assays with NS‐398 (10 μM). EGFR was inhibited by the EGFR tyrosine kinase inhibitor PD153035 (10 nM) and activated using human EGF (10 ng/ml and 5 ng/ml). Results Immunocytochemistry revealed that PAR2 is expressed on mostly basolateral membranes with some expression on apical membrane and punctate immunoreactivity in the cytoplasm in polarized cells. Concentration‐response studies revealed that 2.5 μM 2fLI elicited the highest intracellular Ca 2+ response (EC 50 = 0.5 μM). PAR2 activation at 10 μM 2fLI significantly increased wound healing (P≤ 0.0001). However, PAR2 activation by 2fLI did not induce COX‐2 expression, nor did COX‐2 inhibition alter PAR2‐induced wound healing. PAR2‐induced wound healing was inhibited by the EGFR tyrosine kinase inhibitor (P ≤ 0.001). Moreover, 2fLI induced wound healing is comparable to the that of EGF (5 ng/ml). Conclusion PAR2 activation drives wound healing in CMT93 epithelial cells through the transactivation of EGFR and not through induction of COX‐2. This study enhances our understanding of how proteases in the inflammatory milieu may drive epithelial restitution and mucosal healing. Support or Funding Information 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 .