Stimulation of cAMP production and cyclooxygenase-2 by prostaglandin E2 and selective prostaglandin receptor agonists in murine osteoblastic cells

Prostaglandins (PGs), particularly PGE2, can stimulate bone resorption and formation and auto-amplify their effects by inducing cyclooxygenase (COX)-2. We examined the role of different PG receptors in stimulating cAMP production and COX-2 expression in murine calvarial osteoblasts. Cells were obtained from PGE2 receptor (EP2R and EP4R) wild-type and knockout (KO) mice and from mice transgenic for the COX-2 promoter fused to a luciferase reporter. We analyzed effects of selective agonists, EP2A and EP4A, for EP2R and EP4R, which mediate the increase in cAMP in response to PGE2. We also tested agonists for other PGE2 receptors (EP1A and EP3A) and for prostacyclin (IPA), prostaglandin D2 (DPA), thromboxane (TPA), and prostaglandin F2α (FPA) receptors. PGE2 and EP2A were the most effective stimulators of cAMP production. EP4A, IPA, and DPA produced smaller responses, and EP1A, EP3A, FPA, and TPA were ineffective. In EP2R KO cells, cAMP responses to PGE2 were reduced by 80%, and responses to EP2A were abrogated. In EP4R KO cells, cAMP responses to PGE2 and EP2A showed a small reduction, while the response to EP4A was abrogated. Pretreatment with PGE2, EP2A, or EP4A down-regulated the subsequent response to the respective ligands. COX-2 induction was measured by increased luciferase activity and mRNA expression. PGE2 was the most effective agonist; EP2A and another selective EP2R agonist, butaprost, showed similar efficacy, and EP4A was less effective. EP2A and EP4A effects on luciferase activity were additive, and effects of the combination were similar to PGE2 itself. IPA, TPA, and DPA produced 2- to 6-fold increases in COX-2 expression. FPA was a weak agonist, while EP1A and EP3A were inactive. Treatment with specific inhibitors indicated that PGE2, EP2A, and EP4A induced COX-2 expression largely through protein kinase A (PKA). We conclude that the PG induction of COX-2 in this system generally paralleled effects on cAMP production and was mediated predominantly via the PKA pathway.