Signal Transduction Laboratory, Methodist Research Institute, Indianapolis, IN 46202, USA.
Prostaglandins Other Lipid Mediat. 2010 Feb;91(1-2):18-29. doi: 10.1016/j.prostaglandins.2009.12.002. Epub 2009 Dec 14.
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.
前列腺素 E（2）（PGE（2））在影响肿瘤细胞的生物学行为方面起着关键作用。我们之前的研究表明，PGE（2）通过激活蛋白激酶 A（PKA）II 型刺激人神经胶质瘤细胞生长。本研究旨在进一步阐明 PGE（2）下游激活 PKA 的细胞内途径。用 PGE（2）刺激 U87-MG 神经胶质瘤细胞可使环磷酸腺苷反应元件（CRE）结合蛋白 CREB 在 Ser-133 处磷酸化，并呈剂量和时间依赖性地增加 CREB 驱动的转录。表达干扰 CREB 在 Ser-133 处磷酸化或与 CRE 位点结合的显性 CREB 构建体，明显降低 PGE（2）诱导的 CREB 激活。用 H-89 抑制 PKA 或表达显性负 PKA 构建体可减弱 PGE（2）诱导的 CREB 激活。此外，与用载体处理的细胞相比，抑制 PKA II 型可使 PGE（2）诱导的 CREB 依赖性转录减少 45%。为了研究其他信号通路的参与，用 wortmannin 或 LY294002 预处理 U87-MG 细胞以抑制 PI3-激酶/AKT 途径。两种抑制剂对 PGE（2）诱导的 CREB 磷酸化和转录活性均无影响，表明 PGE（2）以 PI3-激酶/AKT 非依赖性方式激活 CREB。用 PGE（2）（诱导最大 CREB 激活的浓度）或佛波醇刺激 U87-MG 细胞可抑制细胞外信号调节激酶（ERK）磷酸化。用 ERK 抑制剂 PD98059 预处理 U87-MG 细胞，可增强 ERK 抑制并增加 PGE（2）刺激的 CREB 在 Ser-133 处的磷酸化和 CREB 驱动的转录，表明 ERK 抑制有助于 PGE（2）诱导的 CREB 激活。PGE（2）或佛波醇抑制 Raf-1 丝氨酸 338 磷酸化的抑制作用可通过用 H-89 或显性负 PKA 构建体处理细胞而被挽救。此外，11-去氧-PGE（1）刺激 U87-MG 细胞中的 CREB 驱动的转录并刺激细胞生长，而其他 PGE（2）类似物则没有作用。我们的研究结果揭示了一种新的信号通路，即 PGE（2）通过 PKA 信号传递抑制 ERK 并增加 CREB 转录活性。
