J. Golden1, A. Dossa1, P. Kavarian1, K. Goldstein1, H. Ford1, C. Gayer1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction: The secondary bile acid, ursodeoxycholic acid (UDCA), plays a role in intestinal epithelial cell signaling. Previous work in our lab has shown that UDCA promotes enterocyte migration possibly through an epidermal growth factor receptor (EGFR)-dependent pathway. However, the downstream signaling events remain unknown. Cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), are key mediators of enterocyte cell signaling and act on four G-protein coupled receptors (EP1-4). PGE2 has been shown to promote enterocyte migration via EP2 receptor activation and transactivation of EGFR. Therefore, we hypothesized that UDCA promotes enterocyte migration by inducing COX-2, which leads to PGE2 production, and downstream activation of mediators such as PI3K, Akt, and ERK via EP2 receptor signaling.
Methods: Rat intestinal epithelial cells (IEC-6) were treated with UDCA (0.1uM to 400uM) for 12 hours. Expression of COX-2 protein was examined using Western blot. IEC-6 cell migration was measured using a modified wound-healing assay by creating circular holes in cell monolayers and observing them over 6 hours. Cells were treated with or without 200uM UDCA, 10uM EP2 antagonist PF-04418949, 10uM PI3K inhibitor LY294002, 1uM AKT inhibitor IV, or 20uM ERK (MEK) inhibitor PD98059. Statistical analysis was performed using Student’s t-test.
Results: UDCA significantly increased COX-2 expression in a dose-dependent manner at doses greater than or equal to 10uM. UDCA at 200uM increased COX-2 expression 2.7 ± 0.6 fold from control (p<0.05). UDCA at 200uM also increased wound closure 10% ±0.04 from control (p<0.05). Pre-treatment with an EP2 antagonist abrogated UDCA-induced IEC-6 cell migration. Similarly, pre-treatment with inhibitors of PI3K, AKT, or ERK respectively, eliminated UDCA-induced cell migration.
Conclusion: These data suggest a novel role for COX-2 in UDCA-induced stimulation of intestinal epithelial cell migration. The mechanism likely involves induction of COX-2 followed by PGE2 production and downstream signaling via the EP2 receptor, PI3K-Akt-ERK pathway. This may represent a novel pathway to enhance epithelial restitution after injury.