Background and purpose
Prostaglandin E&sub2; (PGE&sub2;) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells. Experimental approach: bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE&sub2; with or without agonists/antagonists of PGE&sub2; receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry. Key
Purpose
Prostaglandin E&sub2; (PGE&sub2;) has been implicated in the regulation of adhesion molecules, leukocyte adhesion and infiltration into inflamed site. However, the underlying mechanism therein involved remains ill-defined. In this study, we explored its cellular mechanism of action in the regulation of the intercellular adhesion molecule-1 (ICAM-1) expression in the brain endothelial cells. Experimental approach: bEnd.3 cells, the murine cerebrovascular endothelial cell line and primary mouse brain endothelial cells were treated with PGE&sub2; with or without agonists/antagonists of PGE&sub2; receptors and associated signalling molecules. ICAM-1 expression, Akt phosphorylation and activity of NF-κB were determined by reverse transcription polymerase chain reaction (RT-PCR), immunoblot analysis, luciferase assay and immunocytochemistry. Key
Results
PGE&sub2; significantly up-regulated the expression of ICAM-1, which was blocked by EP4 antagonist (ONO-AE2-227) and knock-down of EP4. PGE&sub2; effects were mimicked by forskolin, dibutyryl cAMP (dbcAMP) and an exchange protein directly activated by cAMP (Epac) activator (8-Cpt-cAMP) but not a protein kinase A activator (N&sup6;-Bnz-cAMP). PGE&sub2;-induced ICAM-1 expression was reduced by knock-down of Epac1. A PI3K specific inhibitor (LY294002), Akt inhibitor VIII (Akti) and NF-κB inhibitors (Bay-11-7082 and MG-132) attenuated the induction of ICAM-1 by PGE&sub2;. PGE&sub2;, dbcAMP and 8-Cpt-cAMP induced the phosphorylation of Akt, IκB kinase and IκBα and the translocation of p65 to the nucleus and increased NF-κB dependent reporter gene activity, which was diminished by Akti.