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PGE₂ Activates Cementoclastogenesis by Cementoblasts via EP4

¹ Department of Oral and Maxillofacial Pathobiology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan;
² Department of Bone and Joint Disease, Research Institute, National Center for Geriatrics and Gerontology, 36-3 Gengo, Obu, Aichi 474-8522, Japan;
³ Department of Periodontology, Graduate School of Medical and Dental Sciences, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan; and
⁴ Department of Periodontics, School of Dentistry, 1959 NE Pacific, D322-Health Science Center, University of Washington, Seattle, WA 98195-7444, USA

View larger version (30K): [in this window] [in a new window]   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.

View larger version (8K): [in this window] [in a new window]   Figure 2. PGE2 regulated RANKL (A), OPG (B), and IL-6 (C) protein expression levels via EP4 in OCCM-30 cells. EP4 agonist, EP4A; EP4 antagonist, EP4R. After 36 hrs of incubation with PGE2 (100 ng/mL) or the EP4 agonist (1 µM), with or without the EP4 antagonist (1 µM), culture media and cells were collected, and RANKL (cell lysate), OPG, and IL-6 (culture media) protein levels were measured by ELISA. Each result was normalized to total protein levels. RANKL protein level was markedly up-regulated by PGE2 and the EP4 agonist (A). PGE2 and the EP4 agonist significantly down-regulated OPG protein levels (B). IL-6 protein levels were significantly up-regulated by PGE2 (C). The EP4 antagonist eliminated these effects of PGE2. Results are expressed as the mean ± SD of 4 cultures. *p < 0.05 and **p < 0.01 vs. control.

View larger version (61K): [in this window] [in a new window]   Figure 3. PGE2 induced cementoblast-mediated cementclastogenesis via EP4 in a co-culture system of OCCM-30 and bone marrow cells. Number of nuclei, n; EP4 agonist, EP4A; EP4 antagonist, EP4R. PGE2 (100 ng/mL) and the EP4 agonist (1 µM) induced TRAP-positive cells (A,B). Although PGE2 induced multi-nucleated TRAP-positive cells, the EP4 agonist induced only mono-nucleated TRAP-positive cells (B). The EP4 antagonist (1 µM) completely suppressed the appearance of PGE2-induced TRAP-positive cells. PGE2 and the EP4 agonist stimulated pit formation on BD BioCoatTM OsteologicTM Multitest slides (C). Arrows indicate resorption pits. Results are expressed as the mean ± SD of 4 cultures. *p < 0.05 and **p < 0.01 vs. control.

Journal of Dental Research, Vol. 86, No. 10, 974-979 (2007)
DOI: 10.1177/154405910708601011


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