Prostaglandin E2 (PGE2) plays a critical role in influencing the biological behavior of tumor cells. We previously demonstrated that PGE2 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 PGE2 downstream to PKA. Stimulation of U87-MG glioma cells with PGE2 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 PGE2-induced CREB activation. Inhibition of PKA by H-89 or expression of a dominant negative PKA construct attenuated PGE2-induced CREB activation. Moreover, inhibition of PKA type II decreased PGE2-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 PGE2-induced CREB phosphorylation and transcriptional activity, suggesting that PGE2 activates CREB in a PI3-kinase/AKT independent manner. Challenge of U87-MG cells with PGE2, 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 PGE2, suggesting that inhibition of ERK contributes to PGE2-induced CREB activation. Inhibition of ERK by PGE2 or by forskolin was rescued by treatment of cells with H-89 or by the dominant negative PKA construct. Moreover, PGE2 or forskolin inhibited phosphorylation of Raf-1 phosphorylation at Ser-338. Challenge of U87-MG cells with 11-deoxy-PGE1 increased CREB-driven transcription and stimulated cell growth, while other PGE2 analogues had no effect. Together our results reveal a novel signaling pathway whereby PGE2 signals through PKA to inhibit ERK and increase CREB transcriptional activity.
|Original language||English (US)|
|Number of pages||12|
|Journal||Prostaglandins and Other Lipid Mediators|
|State||Published - Feb 2010|
Bibliographical noteFunding Information:
We are grateful to Dr. Stanley McKnight for providing the dominant negative PKA construct and Dr. David Ginty for providing the dominant negative CREB constructs. We thank Dr. Cary Mariash for the critical reading of the manuscript. We thank Dr. Dino Rotondo and Dr. Norrie Wilson for helpful discussions. This work was supported by the Methodist Research Institute Funds to M.T.R.
Copyright 2011 Elsevier B.V., All rights reserved.
- Brain tumors