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020 -CAG STUDENT PRIZE M Lejeune, K Chadee Prostaglandin E2 (PGE2) is one of the important pro inflammatory molecules found in the gut and is produced in excess towards an inflammatory insult. In acute gastrointestinal inflammation, high output PGE2 levels are a hallmark of inflammatory bowel diseases and entero-invasive bacterial/parasitic diseases. The mechanism(s) whereby PGE2 alter epithelial barrier function is not known. In this study, we investigated whether PGE2 plays a role in disrupting epithelial barrier function through alteration of plasma membrane ion channels and tight junctions (TJ). Monolayers of T84 human colonic epithelial cells treated with PGE2 showed a concentration (8nM-1µM) and time-dependent precipitous decrease (5 minutes) in Trans Epithelial Resistance (TER) indicating a loss of epithelial cell integrity. Using highly specific inhibitors we determined that both CFTR and Na+ClK co transporters were responsible for the transcellular mode of decrease in TER. Paracellular changes at the TJ caused by PGE2 was not size selective (14C mannitol flux assays) but allowed the passage of ions as revealed by NaCl dilution potential assays. To determine alterations in the TJ complex, cellular fractionation studies were performed to identify shifts of TJ proteins between the cytoplasm and membrane fractions. Of all the TJ proteins studied, only a significant shift of Claudin 4 from the cytoplasm to the membrane fraction was observed. Using highly specific EP receptor agonists/antagonists we identified that PGE2 coupling only to the high affinity EP4 receptor was responsible for mediating PGE2 induced decrease in TER. Surprisingly, this effect was independent of signaling via the PKA or PI3K pathways based on pharmacologic inhibition studies. However, the levels of active (phosphorylated) protein kinase C alpha/betaII which are critical for the normal assembly of TJ, was significantly decreased in PGE2 treated cells. Taken together, these results have identified that coupling of PGE2 to EP4 receptors inhibits active PKC alpha/betaII isoforms to alter epithelial barrier functions. This novel finding for the first time clearly implicates a central role for PGE2 modulating PKC isoforms in the pathogenesis of various enteric inflammatory conditions.
Gastrointestinal Research Group, Faculty of Medicine, University of Calgary, Calgary, Alberta