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Clodronate Inhibits PGE₂ Production in Compressed Periodontal Ligament Cells

¹ Divisions of Orthodontics and Dentofacial Orthopedics,
² Oral Dysfunction Science, and
³ Dental Pharmacology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan

^* corresponding author, igarashi{at}mail.tains.tohoku.ac.jp

	ABSTRACT

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Periodontal ligament (PDL) cells play an essential role in orthodontic tooth movement. We recently reported that clodronate, a non-N-containing bisphosphonate, strongly inhibited tooth movement in rats, and thus could be a useful adjunct for orthodontic treatment. However, it is not clear how clodronate affects the responses of PDL cells to orthodontic force. In this study, we hypothesized that clodronate prevents the mechanical stress-induced production of prostaglandin E₂ (PGE₂), interleukin-1ß (IL-1ß), and nitric oxide (NO) in human PDL cells. A compressive stimulus caused a striking increase in PGE₂ production, while the responses of IL-1ß and NO were less marked. Clodronate concentration-dependently inhibited the stress-induced production of PGE₂. Clodronate also strongly inhibited stress-induced gene expression for COX-2 and RANKL. These results suggest that the inhibitory effects of clodronate on tooth movement and osteoclasts may be due, at least in part, to the inhibition of COX-2-dependent PGE₂ production and RANKL expression in PDL cells.

KEY WORDS: clodronate • periodontal ligament cell • mechanical stress • prostaglandin E₂

	INTRODUCTION

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Periodontal ligament cells play an essential role in orthodontic tooth movement, and mechanically induced bone resorption is known to be a rate-limiting step (Rygh, 1987; Igarashi et al., 1994). Recently, we demonstrated that clodronate, a non-N-containing bisphosphonate that has been used to treat various metabolic bone diseases associated with excessive bone resorption (Plosker and Goa, 1994; Fleisch, 2000), strongly inhibited bone resorption induced by orthodontic mechanical stress and tooth movement in rats, suggesting that it could be a useful adjunct for orthodontic treatment (Liu et al., 2004). A previous study has demonstrated the direct inhibitory action of clodronate on osteoclastic bone resorption, i.e., clodronate induces apoptosis of osteoclasts through incorporation into the cells (Frith et al., 2001). Although the number of osteoclasts on the pressure side of the periodontal ligament decreased in clodronate-treated animals, the mechanism of action in this process has not yet been determined. The purpose of the present study was to clarify how clodronate affects the responses of periodontal ligament cells to orthodontic force, especially those leading to bone resorption. In this study, we hypothesized that clodronate prevents the mechanical stress-induced production of prostaglandin E₂ (PGE₂), interleukin 1ß (IL-1ß), and nitric oxide (NO) in cultured human periodontal ligament cells, which are known to play important roles in the bone-resorptive responses to orthodontic mechanical stimulation (Yamasaki et al., 1980; Chumbley and Tuncay, 1986; Saito et al., 1991; Zhou et al., 1997; Alhashimi et al., 2001; Iwasaki et al., 2001; Hayashi et al., 2002; Shirazi et al., 2002).

	MATERIALS & METHODS

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The protocol for the experiment was approved by the Research Ethics Committee of Tohoku University Graduate School of Dentistry, and informed consent was obtained from all patients.

Drug
Clodronate (dichloromethylene bisphosphonate disodium salts) was obtained from Procter & Gamble Pharmaceuticals’ Woods Corners Laboratories (Norwich, NY, USA).

Compression of Primary Human Periodontal Ligament Cells
Primary periodontal ligament cells were derived from human tooth roots extracted for orthodontic treatment. Donors were healthy young adults of both sexes (from 20 to 34 yrs old), free of periodontal disease. The cells were cultured in -MEM supplemented with 10% FBS, antibiotics, and 1 x 10^–8 M 1 ,25-dihydroxyvitamin D₃ (Duphar, Amsterdam, Netherlands) at 37°C in an atmosphere of 5% CO₂ in humidified air. The medium was changed every 5 days, and the cells underwent from 4 to 8 passages until use.

For the experiment, periodontal ligament cells were seeded on 35-mm wells in a six-well plate at a density of 3 x 10⁵ cells/dish and cultured until they were confluent. They were then transferred to 2 mL of fresh medium that contained a specific concentration of clodronate and cultured for an additional 24 hrs. After the pre-culture, the cells were continuously compressed according to the method described previously (Kanzaki et al., 2002). Briefly, compressive force was applied directly to periodontal ligament cells by the placement of a custom-made glass cylinder (diameter, 30.3 mm; height, 14.8 mm; thickness, 2.0 mm) that contained lead granules over a confluent cell layer in the well. We adjusted the force magnitude by adding or reducing the granules. In the present study, the cells were subjected to 2.0 g/cm² of compressive force for 48 hrs. After the experiment, total RNA was extracted from each culture with the use of the QuickPrep Total RNA Extraction Kit (Pharmacia Biotech, Uppsala, Sweden). The culture medium was also withdrawn and stored at –20°C for determination of PGE₂, IL-1ß , and NO. The concentrations of PGE₂ and IL-1ß were measured with respective specific enzyme immunoassay kits (for PGE₂, RPN222, Amersham Pharmacia Biotech, Piscataway, NJ, USA; for IL-1ß, QLB00, R&D Systems, Inc., Minneapolis, MN, USA). We evaluated NO production by measuring nitrite and nitrate concentrations in the medium using the HPLC-Griess method (Ohta et al., 1994).

Since responsiveness of cultured human periodontal ligament cells varies depending on their sources, the experiment was repeated, and each single experiment was performed with cells from a different subject.

Semi-quantitative Reverse-transcription Polymerase Chain-reaction (RT-PCR) Assays for Cyclo-oxygenase-2 (COX-2) and Receptor Activator Nuclear Factor B Ligand (RANKL) Gene Expression
We reverse-transcribed extracted RNA to synthesize cDNA using You-Prime First Strand Beads (Pharmacia Biotech) and Oligo (dT)₁₅ primer (Promega, Madison, WI, USA). First-strand cDNA was then subjected to PCR amplification with gene-specific PCR primers. The primers used in this study were: 5'-AGC AGA GAA AGC GAT GGT-3' (forward) and 5'-GGG TAT GAG AAC TTG GGA TT-3' (reverse) for RANKL, 5'-AAC CCA CTC CAA ACA CAG-3' (forward) and 5'-CTG GCC CTC GCT TAT GAT CT-3' (reverse) for COX-2, and 5'-ATG AGG ATC CTC ACC GAG CGC GGC TAC AGC-3' (forward) and 5'-ACA CCA CTG TGT TGG CGT ACA GGT CTT TGC-3' (reverse) for ß-actin. PCR was performed with a KOD Dash DNA Polymerase Kit (Toyobo Co., Ltd.; LDP-101, Tokyo, Japan). Annealing temperatures were 58°C for RANKL, 51°C for COX-2, and 58°C for ß-actin. Numbers of PCR cycles were 42–44 for RANKL, 32–33 for COX-2, and 27 for ß-actin. The PCR products were subjected to electrophoresis and stained with ethidium bromide. The relative intensities of the gel bands were measured with the use of Scion Image Analysis software (Scion Co., Frederick, MD, USA). The method has been described in detail previously (Kanzaki et al., 2002).

Statistical Analysis
The data were subjected to one-way analysis of variance (ANOVA), followed by Fisher’s PLSD test. P < 0.05 was considered a significant difference.

	RESULTS

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Clodronate showed different effects on PGE₂, IL-1ß , and NO production in periodontal ligament cells induced by compressive mechanical stress (Fig. 1). The compression of cells at 2.0 g/cm² for 48 hrs caused nearly a 30-fold increase in PGE₂ release (Fig. 1A), while the increase was not significant for IL-1ß (Fig. 1B) and only minimal for NO (Fig. 1C). Clodronate (5, 25, 125 µM) concentration-dependently inhibited the mechanical stress-induced increase in PGE₂ production in periodontal ligament cells (Fig. 1A). The inhibitory effect of clodronate on NO production was significant only at the highest concentration (125 µM) (Fig. 1C).

View larger version (15K): [in this window] [in a new window]   Figure 1. Effects of clodronate on prostaglandin E2 (PGE2) (A), interleukin 1ß (IL-1ß) (B), and nitric oxide (NO) (C) production in periodontal ligament cells induced by compressive mechanical stress. Each column and bar represent the mean ± SEM (n = 3). *Significant increase vs. control (P < 0.05). **Significant increase vs. control (P < 0.01). P < 0.05 compared with load. P < 0.01 compared with load. CLO: clodronate (5, 25, 125 µM).

View larger version (58K): [in this window] [in a new window]   Figure 2. Effect of clodronate on gene expression for cyclo-oxygenase-2 (COX-2) and receptor activator nuclear factor B ligand (RANKL) in compressed periodontal ligament cells. (A) RT-PCR for COX-2, RANKL, and ß-actin. CLO: 125 µM. (B) Relative expression of RANKL mRNA and COX-2 mRNA determined by densitometric analysis. Values were corrected for ß-actin mRNA expression. Representative results of 1 of 3 independent experiments are shown. CLO: clodronate (5, 25, 125 µM).

View this table: [in this window] [in a new window]   Table. Comparison of the Effects of Clodronate on Stress-induced Prostaglandin E2 (PGE2), Cyclo-oxygenase-2 (COX-2), and Receptor Activator Nuclear Factor B Ligand (RANKL) in Human Periodontal Ligament Cells among Experiments (individuals)

	DISCUSSION

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The present results clearly demonstrated that clodronate could also prevent the mechanical stress-induced production of PGE₂ by periodontal ligament cells, which is one of the most important signaling molecules in the responses of periodontal ligament to orthodontic force (Yamasaki et al., 1980; Saito et al., 1991; Kanzaki et al., 2002). The compressive stimulus caused a striking increase in PGE₂ production, while responses were less marked for IL-1ß and NO. Clodronate significantly inhibited the mechanical stress-induced production of PGE₂ in a concentration-dependent manner. Furthermore, clodronate strongly inhibited stress-induced gene expression for COX-2 and RANKL.

Prostaglandins have been shown to play a crucial role in osteoclast formation induced by orthodontic mechanical stress (Yamasaki et al., 1980; Sandy and Harris, 1984; Zhou et al., 1997). Recently, Kanzaki et al.(2002) demonstrated that compressive force stimulates osteoclastogenesis in the co-culture of peripheral blood mononuclear cells with periodontal ligament cells, by increasing the expression of RANKL in periodontal ligament cells. RANKL is known to be an essential factor in the differentiation and activation of osteoclasts (Suda et al., 1999). It has also been demonstrated that this increase in RANKL expression paralleled that in COX-2 expression and was dependent on PGE₂ production (Kanzaki et al., 2002). Clodronate inhibited all of these responses in compressed periodontal ligament cells, suggesting that it may have decreased RANKL expression in these cells by inhibiting the COX-2-dependent production of PGE₂. At present, the mechanism by which clodronate inhibits COX-2 expression in periodontal ligament cells is not known. Although NO and IL-1 have been shown to induce COX-2 in osteoblastic cells (Buttery et al., 2002; Pilbeam et al., 2002), their involvement is not likely, since the effects of mechanical stress with or without clodronate on the production of these molecules were only minimal or insignificant.

In our previous in vivo study, the number of osteoclasts on the pressure side of the periodontal ligament decreased in clodronate-injected animals (Liu et al., 2004), indicating that clodronate may have either inhibited the recruitment of osteoclasts, promoted osteoclast apoptosis, or both (Rogers et al., 2000). The present in vitro results suggest that clodronate may have impaired the ability of periodontal ligament cells to support osteoclast formation by decreasing RANKL expression. It is also possible that the decreased expression of RANKL promoted osteoclast apoptosis, and hence decreased the number of osteoclasts, since RANKL has been shown to act as a survival factor and to prevent apoptosis of osteoclasts (Lacey et al., 2000). Osteoclast apoptosis has been considered to be a major mechanism of action for the inhibition of bone resorption by this bisphosphonate (Halasy-Nagy et al., 2001). Frith et al.(2001) demonstrated that clodronate is incorporated into osteoclasts and metabolized to adenosine 5'-(ß, -dichloromethylene) triphosphate, which may induce apoptosis in these cells. In addition to the formation of this ATP analogue, the inhibition of RANKL expression in supporting cells like periodontal ligament cells might also be involved in the induction of apoptosis in osteoclasts.

In conclusion, the present results suggest that the inhibitory effects of clodronate on orthodontic tooth movement and osteoclasts may be due in part to the inhibition of COX-2-dependent PGE₂ production, which leads to decreased RANKL expression in periodontal ligament cells subjected to orthodontic mechanical stress.

	ACKNOWLEDGMENTS

 
This research was supported by a Grant-in-Aid for Scientific Research (C) from the Ministry of Education, Science, Sports, and Culture of Japan (No. 13672138).

	FOOTNOTES

 
⁴ present address, Department of Orthodontics, Dalian University Stomatology Hospital, Dalian, China

Received August 8, 2005; Last revision April 23, 2006; Accepted May 3, 2006

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