A70 PROTEASE-ACTIVATED RECEPTOR 2 DRIVES COLONIC EPITHELIAL WOUND HEALING VIA EGFR TRANSACTIVATION
Bibliographic record
Abstract
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 widely present in the gastrointestinal tract and 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. We hypothesized that PAR2 activation drives COX-2 and EGFR dependent epithelial wound healing. 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). The functionality of PAR2 was tested by measuring intracellular Ca2+, since it has been shown that PAR2 activation elicits Ca2+-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 mM). EGFR was inhibited by the EGFR tyrosine kinase inhibitor PD153035 (10 nM) and activated using human EGF (10 ng/ml and 5 ng/ml). 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 mM 2fLI elicited the highest intracellular Ca2+ response (EC50 = 0.5 mM). PAR2 activation at 10 mM 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). 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. CIHR
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How this classification was reachedexpand
Full frame machine prediction
Teacher imitationNot calibrated prevalence, not ground truth. Human validation pending. The Gemma side is a direct model label for every work in the frame, read from the title-only record. The Codex side is a classifier learned from the 10,348 direct Codex labels and calibrated to design-weighted sample rates; fields without enough sample support carry no Codex call. Candidate is the union of the two sides; consensus is their intersection. These outputs are machine_predicted_unvalidated and are not human labels.
Distilled classifier scores by category (both heads)
| Category | Codex | Gemma |
|---|---|---|
| Metaresearch | 0.000 | 0.000 |
| Meta-epidemiology (narrow) | 0.000 | 0.000 |
| Meta-epidemiology (broad) | 0.000 | 0.001 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.001 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.001 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 0.002 |
Machine scores (provisional)
The two teacher heads of the student model, read on this work. A score orders the frame for review; it never asserts a category, and the validation status ships verbatim with every row.
Baseline scores from an immature model (maturity gate not passed, 7 training rounds). Scores rank; they never assert a category.
score_only:v0-immature-baseline · verbatim from the scoring run: score_only means the number may rank works, and no category label ships from itClassification
machine, unvalidatedMachine predicted; a candidate call from one source (direct Gemma or distilled Codex), not a consensus.
How this classification was reached, model by model and score by score, is at the end of the page under "How this classification was reached".