J. Golden1, P. Kavarian1, L. Illingworth1, J. Lim1, J. Wang1, A. Grishin1, H. Ford1 1Children’s Hospital Los Angeles,Pediatric Surgery,Los Angeles, CA, USA
Introduction: Cyclooxygenase-2 (COX-2) and its product, prostaglandin E2 (PGE2), have been identified as critical factors in inflammatory gut barrier failure. PGE2 is known to act on four receptors (EP1-EP4) all of which are present in the intestinal epithelium. Previous work in our lab has shown that high levels of COX-2 and PGE2 cause intestinal barrier breakdown in experimental peritonitis. Therefore, we hypothesized that COX-2 is induced by its end product, PGE2, via EP receptor activation resulting in runaway inflammation.
Methods: Intestinal epithelial cells (IEC-6) were treated for 12 hours with PGE2, PGE2 following EP receptor antagonist pre-treatment, and with PGE2 following pre-treatment with inhibitors of various downstream mediators of the EP1-4 pathways (PKC, PI3K, MEK). Expression of COX-2 protein was examined using Western blot. Results were obtained using quantitative immunofluorescent protein detection and statistical analysis was performed using Student’s t-test.
Results: COX-2 protein levels were significantly increased 2.6 (±0.5) fold from control after treatment with 100uM PGE2. PGE2-induced COX-2 expression was attenuated when cells were pre-treated with EP2 antagonist PF-04418948 and EP4 antagonist GW627368, but not with EP1 or EP3 antagonists. Pre-treatment with a PKC inhibitor, a downstream mediator of EP1, did not attenuate PGE2-induced COX-2 expression. Inhibiting mediators downstream of EP2 and EP4, such as PI3K and MEK, eliminated PGE2-induced COX-2 induction.
Conclusion: COX-2 is induced by its end product PGE2 in IEC-6 cells. This induction is attenuated if cells are pre-treated with EP2 and EP4 antagonists or with inhibitors of their downstream mediators, MEK and PI3K. This attenuation is not seen with EP1 or EP3 antagonists or inhibition of PKC. Our data suggest that PGE2 causes induction of COX-2, likely via an EP2/EP4-dependent pathway, contributing to the runaway inflammatory response seen in inflammatory intestinal disorders.