A62 PROTEASE-ACTIVATED RECEPTOR-2 ACTIVATION ENHANCES EPITHELIAL WOUND HEALING THROUGH SRC PATHWAY-DEPENDENT CELL MIGRATION
Bibliographic record
Abstract
Abstract Background Protease-activated receptors (PARs) and their activating enzymes play a role in inflammatory bowel disease (IBD) pathogenesis, but the specific roles of PAR2 in disease initiation and progression remain unclear. Interestingly, PAR2 activation has both pro-proliferative and pro-migratory effects and could be involved with restoration of the epithelial barrier following injury. We previously showed that the PAR2 activation increase the epithelial wound healing through ERK, PI3K and JNK pathways. However, the role of Src kinase, which is also activated by PAR2, is not known. Aims We hypothesized that PAR-2 activation induces a wound healing response in intestinal epithelial cells through Src activity. Methods Circular wounds were made in T84 colonic epithelial cells monolayers. Wounded monolayers were treated with the PAR2 activating peptide, 2-furoyl-LIGRLO (2fLI, 5 μM), or the inactive control reverse-sequence peptide, 2-furoyl-OLRGIL (2fO, 5 μM), and live cell imaging was used to record wound closure over a 12 or 24-hr period. The specificity of PAR2 was assessed with a PAR2 inhibitor (GB88). Proliferation and cytotoxicity were measured using EdU and TUNEL assays. The mechanism of action was evaluated using inhibitors of FAK (PF57328 and FAK14) and Src (PP2) and western blot (WB) was used to confirm the protein levels [p-FAK (Y397; Y576/577) and p-Src (Y416)]. For immunofluorescence, images of E-cadherin and F-actin or p-FAK were taken to capture the entire wound border and surrounding cells. Results PAR2 activation by 2fLI promoted wound closure compared to 2fO or vehicle control at the 12 and 24 hr timepoints ( p<0.05). PAR2’s ability to enhance wound closure was blocked with the PAR2 inhibitor, GB88 ( p<0.01). PAR2 activation had no effect on proliferation at the wound-edge and did not cause apoptosis, but enhanced lamellipodia/filopodia formation ( p<0.001), indicating that PAR2 might program the cells toward a migratory phenotype rather than a proliferative phenotype. When we investigated the mechanisms of action, PAR2 activation did not induce focal adhesion kinase (FAK) expression in T84 cells and inhibition of FAK by selective inhibitors (PF573288 and FAK14) did not alter PAR2-induced wound healing. The immunofluorescence for p-FAK (Y397) and WB (Y937 and Y576/577) confirmed these results. However, the Src tyrosine kinase inhibitor (PP2, p<0.0001) inhibited PAR2-induced wound healing. PAR2 activation increased Src phosphorylation (Y416, p<0.05). Conclusions PAR2 activation drives wound healing in part via Src tyrosine kinase activity, but independently of FAK activity. These findings provide a further mechanism whereby PAR2 can participate in the resolution of intestinal wounds in gastrointestinal inflammatory diseases. Funding Agencies CAG, 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 | 0.000 |
| Bibliometrics | 0.000 | 0.000 |
| Science and technology studies | 0.000 | 0.000 |
| Scholarly communication | 0.000 | 0.000 |
| Open science | 0.000 | 0.000 |
| Research integrity | 0.000 | 0.001 |
| Insufficient payload (model declined to judge) | 0.004 | 0.001 |
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".