Fig. 2.

The EP4 receptor is responsible for PGE₂-mediated induction of Id1. (A) Treatment of primary mouse glioblastoma (GBM) cultures or human U87, LN-229, or U118 glioblastoma cells with 0.5 µM dmPGE₂ led to time-dependent induction of Id1 protein. (B) GBMs were initiated in Nestin-tva, Arf ^−/− mice at day zero via stereotactic injection RCAS-PDGFB virus. At day 23, tumor-bearing mice were treated with 100 mg/kg/day celecoxib or vehicle control (n = 4 per group) by gavage for 4 days, followed by sacrifice and tumor harvest. Harvested tissue was subjected to mass spectrometry to quantify PGE₂ levels in normal brain versus GBM with and without celecoxib treatment. *, P < .05, Error bars represent mean ± SEM. (n = 4). (C) Immunohistological analysis of Id1 in tumors derived from mice treated with vehicle (n = 4) or celecoxib (n = 3). Three non–overlapping fields were quantitated for each tumor. Scale bar = 0.1 mm. Error bars represent mean ± SEM. (n = 3 or 4). *, P < .05. (D) To identify the relevant PGE₂ receptor in GBM cells, we performed quantitative real-time (qRT) PCR for the 4 EP receptors in mouse GBM cells (upper panel) and human U87 cells (bottom panel). The level of EP4 was notably higher than EP1-3, when EP1 was used as the point of comparison in both cell types, while U87 cells also displayed elevated EP2. Error bars represent mean ± SEM. (n = 3). ***, P < .001. (E) EP2/4 receptor expression was knocked down in human U87 GBM cells via transient transfection with specific siRNAs. Id1 upregulation in response to dmPGE₂ treatment was inhibited specifically with EP4 knockdown. (F) Treatment of primary mouse GBM cells or human LN-229 or U118 glioblastoma cell lines with the EP4 receptor agonist PGE1-alcohol resulted in a dose-dependent increase in Id1 protein levels. (G) Treatment with an EP4 antagonist (AH23848) effectively blocked dmPGE₂-mediated induction of Id1.