Prostaglandin E(2) (PGE(2)) plays a critical role in influencing the biological behavior of tumor cells. We previously demonstrated that PGE(2) stimulates human glioma cell growth via activation of protein kinase A (PKA) type II. This study was undertaken to further elucidate the intracellular pathways activated by PGE(2) downstream to PKA. Stimulation of U87-MG glioma cells with PGE(2) increased phosphorylation of the cyclic-AMP response element (CRE) binding protein CREB at Ser-133 and CREB-driven transcription in a dose- and time-dependent manner. Expression of dominant CREB constructs that interfere with CREB phosphorylation at Ser-133 or with its binding to the CRE site markedly decreased PGE(2)-induced CREB activation. Inhibition of PKA by H-89 or expression of a dominant negative PKA construct attenuated PGE(2)-induced CREB activation. Moreover, inhibition of PKA type II decreased PGE(2)-induced CREB-dependent transcription by 45% compared to vehicle-treated cells. To investigate the involvement of additional signaling pathways, U87-MG cells were pretreated with wortmannin or LY294002 to inhibit the PI3-kinase/AKT pathway. Both inhibitors had no effect on PGE(2)-induced CREB phosphorylation and transcriptional activity, suggesting that PGE(2) activates CREB in a PI3-kinase/AKT independent manner. Challenge of U87-MG cells with PGE(2), at concentrations that induced maximal CREB activation, or with forskolin inhibited extracellular signal-regulated kinase (ERK) phosphorylation. Pretreatment of U87-MG cells with the ERK inhibitor PD98059, accentuated ERK inhibition and increased CREB phosphorylation at Ser-133 and CREB-driven transcription stimulated by PGE(2), suggesting that inhibition of ERK contributes to PGE(2)-induced CREB activation. Inhibition of ERK by PGE(2) or by forskolin was rescued by treatment of cells with H-89 or by the dominant negative PKA construct. Moreover, PGE(2) or forskolin inhibited phosphorylation of Raf-1 phosphorylation at Ser-338. Challenge of U87-MG cells with 11-deoxy-PGE(1) increased CREB-driven transcription and stimulated cell growth, while other PGE(2) analogues had no effect. Together our results reveal a novel signaling pathway whereby PGE(2) signals through PKA to inhibit ERK and increase CREB transcriptional activity.