Figure 1. Effect of PGE₂ on the expression of RANKL, OPG, M-CSF, IL-6, and COX-2 mRNA (A) time-course and the role of PGE receptor subtypes (EPs) on RANKL (B,C), OPG (D,E), and IL-6 (F,G) mRNA expression 2 hrs after PGE₂ stimulation in OCCM-30 cells. EP1 agonist, EP1A; EP2 agonist, EP2A; EP3 agonist, EP3A; EP4 agonist, EP4A; EP1 antagonist, EP1R; EP2 antagonist, EP2R; EP3 antagonist, EP3R; EP4 antagonist, EP4R. (A) OCCM-30 cells were treated with PGE₂ (100 ng/mL) for 0–24 hrs. RANKL, IL-6, and COX-2 mRNA expression levels were increased at 2 hrs with PGE₂ stimulation. Cementoblasts were exposed to PGE₂ (100 ng/mL) or EP agonist (1 µM) for 2 hrs, and expression of RANKL, OPG, and IL-6 mRNAs in OCCM-30 cells was analyzed by quantitative RT-PCR. To eliminate the effects of endogenous PGE₂, we pre-treated the cells with NS-398 (5 µM). PGE₂ and EP4 agonist significantly up-regulated RANKL mRNA expression (B). The effect of PGE₂ on RANKL mRNA expression was eliminated by the EP4 antagonist (1 µM) (C). The EP4 agonist strongly suppressed OPG mRNA expression, while PGE₂ and other EP agonists slightly suppressed OPG mRNA expression (D). The EP4 antagonist (1 µM) eliminated the suppressive effect of PGE₂ on OPG mRNA, and, in fact, up-regulated the expression of OPG mRNA (E). IL-6 mRNA expression was drastically enhanced by PGE₂ (F), and this effect was eliminated with the EP4 antagonist (1 µM) (G). Results are expressed as the mean ± SD of 4 cultures. *p < 0.05 and **p < 0.01 vs. control; ##p < 0.01 between 2 experimental groups.