Brain natriuretic peptide (BNP) produced by cardiac myocytes has anti-fibrotic and anti-growth properties and is a marker of cardiac hypertrophy. We showed that prostaglandin E₂ (PGE₂) is the main prostaglandin produced in myocytes treated with pro-inflammatory stimuli and stimulates protein synthesis by binding to its EP₄ receptor. We hypothesized that PGE₂, acting through EP₄, also regulates BNP gene expression. We transfected neonatal ventricular myocytes with a plasmid encoding the human BNP (hBNP) promoter driving expression of a luciferase reporter gene. PGE₂ increased hBNP promoter activity 3.5-fold. An EP₄ antagonist reduced the stimulatory effect of PGE₂ but not an EP₁ antagonist. Since EP₄ signaling can involve adenylate cyclase, cAMP and protein kinase A (PKA), we tested the effect of H-89, a PKA inhibitor, on PGE₂ stimulation of the hBNP promoter. 5 µM H-89 decreased PGE₂ stimulation of BNP promoter activity by 100%. Since p42/44 MAPK mediates PGE₂'s effect on protein synthesis, we also examined MAPKs in the regulation of BNP promoter activity. PGE₂ stimulation of the hBNP promoter was inhibited by a MEK1/2 inhibitor and a dominant-negative mutant of Raf, indicating that p42/44 MAPK was involved. In contrast, neither a p38 MAPK inhibitor nor a JNK inhibitor reduced the stimulatory effects of PGE₂. Involvement of small GTPases was also studied. Dominant-negative Rap inhibited PGE₂ stimulation of the hBNP promoter, but dominant-negative Ras did not. We concluded that PGE2 stimulates the BNP promoter mainly via EP₄, PKA, Rap and p42/44 MAPK.