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PGE2 Activates Cementoclastogenesis by Cementoblasts via EP4
H. Oka1,
M. Miyauchi1,*,
K. Sakamoto1,
S. Moriwaki2,
S. Niida2,
K. Noguchi3,
M.J. Somerman4 and
T. Takata1,*
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
Correspondence: * corresponding authors, mmiya{at}hiroshima-u.ac.jp and ttakata{at}hiroshima-u.ac.jp
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ABSTRACT
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Destruction of cementum and alveolar bone is the main causative event for the exfoliation of teeth as a consequence of periodontitis. Prostaglandin E2 (PGE2) and PGE receptor subtypes (EPs) play an important role in modulating osteoblast-mediated osteoclastogenesis; however, no information is available on the role of PGE2 and EPs in regulating cementoblast-mediated cementoclastogenesis. We hypothesized that the PGE2-EPs pathway also regulates cementoblasts’ ability to activate cementoclasts. For these studies, OCCM-30 cells (a mouse cementoblast cell line) were exposed to PGE2 and specific EP agonists. PGE2 (100 ng/mL) and EP4 agonist (1 µM) up-regulated RANKL and IL-6 mRNA levels, while they down-regulated OPG mRNA expression. The EP4 antagonist (1 µM) eliminated these effects of PGE2. PGE2 treatment of co-cultures of OCCM-30 cells with bone marrow cells induced TRAP-positive cells via the EP4 pathway. These findings suggest that PGE2 promotes cementoblast-mediated cementoclastogenesis by regulating the expression of RANKL and OPG via the EP4 pathway.
Key Words: prostaglandin E2 • PGE receptor subtypes • cementoblasts • periodontal tissue
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INTRODUCTION
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Periodontitis is an infectious disease associated with the destruction of alveolar bone, cementum, and periodontal ligament (Freeman, 1994; Carranza and Bernard, 2002). Cementum, a thin mineralized tissue covering the tooth root surface, assists in anchoring teeth to surrounding alveolar bone. Cementoblasts share many characteristics with osteoblasts. There is a possibility that cementoblasts may contribute to not only cementum formation but also cementum destruction, in the same manner as do osteoblasts. However, little is known about the mechanisms controlling cementoclastogenesis by cementoblasts.
It is generally accepted that a variety of products, including prostaglandin E2 (PGE2) and cytokines, produced by host cells may contribute to periodontal tissue destruction. Previously, we demonstrated that osteoblasts and cementoblasts constitutively express cyclooxygenase-2 (COX-2), a PGE2-synthesizing enzyme, and that the immunoexpression of COX-2 in both cell types was increased in rat periodontal tissue after the topical application of LPS (Miyauchi et al., 2004). These findings suggest that PGE2 from these cells may be associated with the destruction of alveolar bone and cementum with LPS-induced periodontitis. PGE2, a major eicosanoid produced by osteoblasts, is one of the most important regulators of bone metabolism (Raisz et al., 1979; Yokota et al., 1986). Moreover, it has been well-established that PGE2 regulates osteoclast formation via effects on the receptor activator of the NF B ligand (RANKL)/osteoprotegerin (OPG) system in osteoblasts (Suda et al., 2004; Choi et al., 2005). PGE2 can also increase the production of IL-6, a known promoter of bone destruction. Various biological actions of PGE2 are mediated by PGE-specific G-protein-coupled receptors (EP), namely, EP1, EP2, EP3, and EP4 (Namba et al., 1993; Coleman et al., 1994; Negishi et al., 1995; Narumiya et al., 1999). Recently, it was reported that PGE2 induced osteoclast formation via the EP4 signaling pathway (Sakuma et al., 2000; Suzawa et al., 2000). The PGE2-controlled RANKL/OPG system seems to operate in cementoblasts like osteoblasts. Therefore, we hypothesized that PGE2 plays a critical role via EPs in the destruction of cementum by promoting cementoblast-mediated cementoclastogenesis under inflammatory conditions.
For the studies here, we focused on cementoclastogenesis by cemenoblasts and determined whether exposure of cementoblasts to PGE2 and specific EP agonists would promote the expression of mRNA for osteoclastogenesis-related factors, including RANKL, OPG, and IL-6. We used a co-culture system of OCCM cells and bone marrow cells to determine the ability of PGE2 to promote the formation of TRAP-positive cells.
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MATERIALS & METHODS
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Cell Line and Cell Culture
OCCM-30 cells, a subclone of the osteotag mouse-derived immortalized cementoblast cell line (D’Errico et al., 1999, 2000), were maintained in alpha-Minimum Essential Medium ( -EM) (Invitrogen Corporation, Carlsbad, CA, USA) with 10 mM HEPES buffer (pH 7.2), 10% FBS, and 100 U/mL penicillin-streptomycin at 37°C in a humidified atmosphere of 5% CO2.
Reagents
PGE2 was purchased from Advanced Magnetics Inc. (Cambridge, MA, USA). ONO-DI-004 (EP1 agonist), ONO-AE1-259-01 (EP2 agonist), ONO-AE-248 (EP3 agonist), ONO-AE1-329 (EP4 agonist), ONO-8713 (selective EP1 antagonist), ONO-AE3-240 (selective EP3 antagonist), and ONO-AE3-208 (selective EP4 antagonist) were kindly provided from ONO Pharmaceuticals Co. Ltd. (Tokyo, Japan). AH6809 (EP2 antagonist) and NS-398 were purchased from Cayman Chemical (Ann Arbor, MI, USA). Mouse M-CSF and ELISA kits of mouse RANKL, OPG, and IL-6 were purchased from R&D Systems (Minneapolis, MN, USA).
RNA Extraction and RT-PCR Analysis
OCCM-30 cells were seeded in 60-mm culture dishes (1 x 106 cells/dish) and cultured in -MEM containing 10% FBS for 2 days. After incubation in -MEM containing 2% FBS for 1 day, the cells were incubated with PGE2 (100 ng/mL). After treatment for 0, 2, 4, 6, 8, 12, and 24 hrs, total RNA was extracted from the cultured cells with TRIzol® Reagent (Invitrogen). cDNAs were synthesized from 1 µg of total RNA with Rever Tra Dash (TOYOBO CO., LTD., Osaka, Japan). Aliquots of total cDNA were amplified with KOD-Plus-DNA Polymerase (TOYOBO CO.). The amplification was performed in a MyCyclerTM thermal cycler (BIO-RAD, Tokyo, Japan). Primer pairs used for PCR, annealing temperatures, and reaction cycles are listed in the Table . PCR products underwent electrophoresis on 1.5% agarose gels at 100 mV and were visualized by ethidium bromide.
Quantitative Real-time RT-PCR Analysis
The cells cultured as described above were pre-treated with NS-398 (5 µM) for 2 hrs to eliminate the effects of endogenous PGE2, and then were incubated with PGE2 (100 ng/mL) or each EP agonist (1 µM). To determine the effects of EP antagonists, we pre-treated the cells with NS-398 and each EP antagonist (1 µM) for 2 hrs prior to the addition of PGE2 (100 ng/mL). After 2 hrs of incubation with PGE2 or each EP agonist, total RNA was extracted, and cDNA was synthesized. Real-time PCR was performed in the Light Cycler System (light cycler quick system 350S, Roche Diagnostics GmbH, Mannheim, Germany) with LightCycler-FastStart DNA Master SYBR Green I (Roche Diagnostics GmbH) and specific primers for RANKL, OPG, and IL-6. The primer pairs and annealing temperatures used are listed in the Table . Reaction product was quantified with GAPDH as the reference gene.
Enzyme-linked Immunosorbent Assay (ELISA)
OCCM-30 cells were seeded in 24-well culture plates (1 x 105 cells/well) and cultured as described above. After 2 hrs of pre-treatment with NS-398 (5 µM), the cells were incubated with PGE2 (100 ng/mL) or EP4 agonist (1 µM). Some cultured cells were pre-treated with NS-398 and EP4 antagonist (1 µM) for 2 hrs before PGE2 stimulation. After 36 hrs of incubation with PGE2 or EP4 agonist, culture media and cells were collected separately for ELISA.
OPG and IL-6 protein levels in culture media and the RANKL protein level in cell lysate, prepared by the Boabaid method (Boabaid et al., 2004), were measured by ELISA as recommended by the manufacturer. Results were normalized to total protein levels. The protein concentration was determined by Bradford protein assay (Bio-Rad, Richmond, CA, USA) with BSA (Sigma) as a standard.
Osteoclast Formation Assay
Bone marrow cells were collected from the femora and tibiae of 6-to 8-week-old male DDY mice. Bone marrow cells (2 x 105 cells/well) were cultured with 20 ng/mL mouse M-CSF in -MEM containing 10% FBS in 96-well culture plates for 2 days to induce the osteoclast precursor cells. OCCM-30 cells (1 x 102 cells/well) and adherent bone marrow cells were then co-cultured for 8 days. Co-cultures were incubated in the presence of PGE2 (100 ng/mL) or EP4 agonist (1 µM) for the final 7 days. Some co-cultures were pre-treated with NS-398 (5 µM) or EP4 antagonist (1 µM) for 2 hrs prior to the addition of PGE2. Co-cultures were fixed and stained for tartrate-resistant acid phosphatase activity (TRAP; a marker enzyme of osteoclasts). All TRAP-positive cells with more than 3 nuclei or with 1 or 2 nuclei appearing in each well were separately counted under a microscope.
Pit Formation Assay
Bone marrow cells and OCCM-30 cells were co-cultured under the conditions described above on BD BioCoatTM OsteologicTM Multitest slides, which consisted of submicron synthetic calcium phosphate thin films coated onto various culture vessels (Becton Dickinson & Co., Bedford, MA, USA). The cells were removed by 6% (w/v) NaOCl and 5.2% (w/v) NaCl, and the resorption pits formed in each vessel were observed under a microscope.
Statistical Analysis
Data are expressed as means ± standard deviation (SD) for each group. Data were subjected to one-factor analysis of variance (ANOVA). Fisher’s protected least significance test was used in the post hoc comparison of specific groups. Probabilities less than 0.05 were considered significant. Each experiment was performed 4 times, with comparable results.
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RESULTS
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PGE2 Regulates RANKL, IL-6, and COX-2 gene Expression in OCCM-30 Cells (Fig. 1A)
PGE2 stimulation increased mRNA expression of RANKL, COX-2, and IL-6 within 2 hrs. Although increased expression was maintained for 24 hrs, expression levels decreased with time.
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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.
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PGE2 Regulates RANKL, OPG, and IL-6 Expression via EP4 in OCCM-30 Cell mRNA Levels (Figs. 1B-1G)
Treatment of OCCM-30 cells with PGE2 (100 ng/mL) and the EP4 agonist (1 µM) significantly increased RANKL mRNA levels at 2 hrs (Fig. 1B). Treatment with only the EP4 antagonist (1 µM) eliminated the stimulatory effect of PGE2 in RANKL mRNA expression (Fig. 1C). EP1, EP2, and EP3 agonists (1 µM) and their EP antagonists (1 µM) had no effect on the regulation of RANKL mRNA expression (Figs. 1B, 1C).
The expression of OPG mRNA in OCCM-30 cells at 2 hrs was down-regulated by PGE2 and each EP agonist (Fig. 1D). The EP4 agonist was the most effective in decreasing OPG mRNA levels. The EP4 antagonist completely eliminated the inhibitory effect of PGE2 on OPG mRNA expression and, in fact, up-regulated OPG transcripts when compared with the control (Fig. 1E).
The expression of IL-6 mRNA in OCCM-30 cells was also enhanced by stimulation with PGE2 for 2 hrs (Fig. 1F), and the stimulatory effect of PGE2 was eliminated completely by treatment with the EP4 antagonist (Fig. 1G). Treatment of cells with the EP4 agonist tended to increase IL-6 mRNA expression, but there was no significant difference between control cells and EP4-agonist-treated cells (Fig. 1F). Other EP antagonists partially, but significantly, reduced PGE2-stimulated IL-6 mRNA expression (Fig. 1G).
Protein Levels (Fig. 2)
PGE2 (100 ng/mL) induced up-regulation of RANKL and IL-6 protein expression in OCCM-30 cells 36 hrs after treatment, and this up-regulation was blocked completely by the addition of the EP4 antagonist (1 µM) (Figs. 2A, 2C). Treatment of cells with the EP4 agonist (1 µM) tended to increase RANKL and IL-6 protein level concentrations measured at 36 hrs, but no significant differences were noted (Figs. 2A, 2C). The level of OPG protein was suppressed significantly with PGE2 or the EP4 agonist (Fig. 2B). This inhibitory effect was eliminated with EP4 antagonist treatment; in fact, the OPG protein level in conditioned medium was significantly higher than that of the control (Fig. 2B).
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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.
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Blocking endogenous PGE2 production with NS-398 did not result in any significant effects on the levels of RANKL, OPG, and IL-6 mRNA and protein expression after either PGE2 or each EP agonist stimulation (data not shown).
PGE2-induced TRAP-positive Cell Formation is Regulated via EP4 in OCCM-30 Cells (Fig. 3)
PGE2 (100 ng/mL) and the EP4 agonist (1 µM) stimulated TRAP-positive cell formation in the co-culture system of OCCM-30 cells and bone marrow cells (Figs. 3A, 3B). The EP4 antagonist (1 µM) completely suppressed the number of TRAP-positive cells induced by PGE2. Although TRAP-positive cells induced with PGE2 contained many large multinucleated cells, the EP4 agonist induced mainly small TRAP-positive mononuclear cells (Fig. 3B). Resorption pits were observed microscopically on OsteologicTM Multitest slides with PGE2 and EP4 agonist stimulation (Fig. 3C).
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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.
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DISCUSSION
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Cementoblasts share many characteristics with osteoblasts. In vivo and in vitro experiments provide strong evidence that PGE2 is involved in the induction of osteoclast-mediated bone resorption (Klein and Raisz, 1970; Raisz, 1982; Akatsu et al., 1989; Shinar and Rodan, 1990; Collins and Chambers, 1991; Wani et al., 1999). PGE2 is considered to induce osteoclast formation by regulating RANKL/OPG expression in osteoblasts via the EP4 pathway (Li et al., 2002). Moreover, the EP4-mediated signaling pathway has been shown to be particularly important for the induction of bone resorption via pro-inflammatory cytokines such as IL-6 (Li et al., 2002; Liu et al., 2005).
In this study, we demonstrated that PGE2 and the EP4 agonist caused an up-regulation of RANKL and IL-6 expression and a down-regulation of OPG expression in cementoblasts. Furthermore, the EP4 antagonist eliminated PGE2 effects on cementoblasts. Analysis of these data suggested that PGE2 regulated RANKL, IL-6, and OPG expression mainly via EP4. In previous reports, using a co-culture of OCCM-30 cells with RAW cells (mouse monocyte macrophage cell line), Boabaid et al.(2004) reported that PTH-related protein (PTHrP) promoted TRAP-positive cell formation via regulating RANKL/OPG levels in cementoblasts. In osteoblasts, PTHrP stimulates RANKL expression and inhibits OPG expression via the cAMP/protein kinase A (PKA) pathway (Lee and Lorenzo, 2002). It is well-known that stimulation of EP4 causes an up-regulation of the cAMP/PKA system (Negishi et al., 1995; Chen and Hughes-Fulford, 2000; Fujino et al., 2002). It is reasonable to consider that PGE2 may induce cementoclast formation by controlling the balance of RANKL/OPG expression levels in cementoblasts via the EP4-cAMP-PKA pathway, in a manner similar to that of PTHrP.
Interestingly, blocking the EP4 pathway by the EP4 antagonist eliminated the ability of PGE2 to down-regulate transcripts for OPG, and, in fact, up-regulated OPG levels in cementoblasts, compared with control cells. Moreover, only EP4 antagonist treatment up-regulated basal levels of OPG mRNA compared with that of untreated control cells (data not shown). There are two possible explanations for the unexpected up-regulation of OPG mRNA caused by EP4 antagonist treatment. One is that there is cross-talk between the IL-6 and PGE2 signaling pathways. Liu et al.(2005) reported that cross-talk between the PGE2 and IL-6 signaling pathways enhanced osteoclast differentiation via effects on the OPG/RANKL/RANK system in bone cells. They reported that treatment of bone cells with IL-6 antibodies promoted an up-regulation of basal OPG mRNA and protein levels, and further eliminated the inhibitory effect of PGE2 on OPG expression. In the present study, the EP4 antagonist completely blocked PGE2-mediated IL-6 production. These results suggest that the inhibition of endogenous IL-6 production mediated by EP4 may be responsible for up-regulation of the basal OPG mRNA level. Another possible explanation is that there is a cAMP-PKA independent pathway associated with the EP4 receptor, which inhibits OPG transcription caused by PGE2 in cementoblasts. Recently, it was reported that PGE2-mediated down-regulation of pro-inflammatory cytokines in macrophages was enhanced via a novel EP4-associated protein in a cAMP-independent manner (Takayama et al., 2006). Such a pathway may be responsible for the up-regulation of OPG with EP4 antagonist treatment.
In the present study, PGE2 induced TRAP-positive cells in a cementoblast/bone marrow cell co-culture system through the EP4 pathway, and those cells demonstrated pit formation activity. Although PGE2 stimulation induced multi-nucleated TRAP-positive cells, EP4 agonist stimulation induced only mono-nucleated TRAP-positive cells. These findings suggest that EP4-pathway-mediated events are necessary to initiate osteoclastogenesis, while alternative pathways may be required for the maturation of multinucleated osteoclasts.
In conclusion, we provide evidence that PGE2 stimulates cementoblast-mediated cementoclast activity in vitro through control of RANKL, IL-6, and OPG mRNA and protein in cementoblasts, mainly via the EP4 pathway, similarly to the role of PGE2 in osteoblasts. Further studies are needed to define the specific signaling pathways controlling PGE2-mediated cementoclastogenesis. Such studies will provide important information toward designing therapies to control the tissue destruction associated with periodontal disease and other inflammatory diseases.
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ACKNOWLEDGMENTS
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This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan.
Received for publication September 6, 2006.
Revision received May 31, 2007.
Accepted for publication June 4, 2007.
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Journal of Dental Research, Vol. 86, No. 10,
974-979 (2007)
DOI: 10.1177/154405910708601011
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ABSTRACT
INTRODUCTION