Neuropeptides modulate RANKL and OPG expression in human periodontal ligament cells

orthodontic waves 66 (2007) 33–40

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Research paper

Neuropeptides modulate RANKL and OPG expression in human periodontal ligament cells Kayoko Nakao a, Tetsuya Goto b,*, Kaori Gunjigake a, Tetsuro Konoo c, Shigeru Kobayashi b, Kazunori Yamaguchi a a

Division of Orofacial Functions and Orthodontics, Kyushu Dental College, Japan Division of Anatomy, Kyushu, Kyushu Dental College, 2-6-1 Manazuru, Kokurakita-ku, Kitakyushu 803-8580, Japan c Division of Comprehensive Dentistry, Kyushu Dental College, Japan b

article info

abstract

Article history:

Neuropeptides, such as substance P (SP) and calcitonin gene-related peptide (CGRP), may be

Received 12 December 2006

associated with bone remodeling in response to mechanical stress during orthodontic tooth

Received in revised form

movement. To investigate this hypothesis, we examined the effects of neuropeptides on the

26 February 2007

expression of receptor activator of nuclear factor kB ligand (RANKL) and osteoprotegerin

Accepted 16 March 2007

(OPG) in human periodontal ligament (PDL) cells under compression in vitro. PDL cells were

Published on line 27 April 2007

subjected to compressive force (2.0 g/cm2) continuously in the presence or absence of SP or CGRP for 2–4 days. The expression of the SP receptor, neurokinin 1-receptor (NK1-R), in PDL

Keywords:

cells was confirmed by RT–PCR and immunofluorescent staining. The effects of neuropep-

Substance P

tides (SP and CGRP) on the expression of RANKL and OPG mRNA were determined using RT–

CGRP

PCR. PDL cells constitutively expressed NK1-R on both the mRNA and protein levels.

RANKL

Compressive force decreased OPG mRNA expression and increased RANKL mRNA expres-

OPG

sion. In the presence of neuropeptides, the OPG level decreased synergistically with

Periodontal ligament cell

compression. Neuropeptides stimulated RANKL expression without compression, whereas they decreased RANKL mRNA expression with compression. These results indicate that PDL cell compression induces the up-regulation of RANKL and down-regulation of OPG, whereas neuropeptides suppress the RANKL expression induced by compression. Therefore, the neuropeptides SP and CGRP may modulate bone remodeling by PDL cells during orthodontic tooth movement. # 2007 Elsevier Ltd and the Japanese Orthodontic Society. All rights reserved.

1.

Introduction

Patients feel dull pain when teeth are moved by orthodontic force. Therefore, a relationship between orthodontic tooth movement and neuropeptides from sensory nerve endings is thought to exist. Sensory neuropeptides, such as substance P

(SP) and calcitonin gene-related peptides (CGRP), are localized in thin, nonmyelinated afferent nerve fibers, transported to peripheral endings, and released by axon reflexes [1]. At the periphery, SP is co-localized with CGRP, and synthesized in the dorsal root ganglion (DRG) or trigeminal ganglion (TG) as a result of nerve-end stimulation [2]. Although sensory

* Corresponding author. Tel.: +81 93 582 1131x6643; fax: +81 93 591 8199. E-mail address: [email protected] (T. Goto). 1344-0241/$ – see front matter # 2007 Elsevier Ltd and the Japanese Orthodontic Society. All rights reserved. doi:10.1016/j.odw.2007.03.004

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neuropeptides have been implicated in inflammatory responses such as vasodilatation and plasma extravasation [3], they also modulate osteoblastic bone formation and osteoclastic bone resorption [4–6]. Tachykinins are widely distributed neuropeptides, particularly in the central nervous system [2,7]. SP is a member of the tachykinin family, and is considered an important neuropeptide in nociceptive processes that works as a neurotransmitter or neuromodulater [8,9]. SP directly modulates bone metabolism through neurokinin-1 receptors (NK1-Rs; SP receptors) [4,6,10–12]. Nerve fibers containing SP are present in the pulp, periodontal ligaments (PDL), and gingiva in rats, cats, and ferrets [13–16]. Moreover, there is an increase in the number of nerve fibers showing CGRP- or SPlike immunoactivity in the periodontal ligament during orthodontic tooth movement [17,18]. On the other hand, previous studies have reported that CGRP inhibit osteoclastic bone resorption and osteoclast differentiation [19–20], although they are much less potent than calcitonin [21]. These findings suggest the relationship between sensory neropeptides and, however, it has not been known whether not PDL cells are affected by neuroprptides through their receptors. Periodontal ligament (PDL) cells initiate bone remodeling in response to the load applied during orthodontic tooth movement [22]. On the compression side, osteoclasts resorb bone. Osteoclast formation and differentiation are regulated by a balance between receptor activator of nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) [23]. PDL cells express both RANKL and OPG [24–27]. However, the relationships between RANKL or OPG expression in PDL cells and the sensory neuropeptides during orthodontic tooth movement are not clear. We then investigated the effects of the sensory neuropeptides SP and CGRP on the expression of RANKL and OPG mRNA in cultured human PDL cells under compression in vitro.

2.2.

Periodontal ligament cells were seeded at 5.0  104 cells per round cover glass (22 mm diameter; Matsunami Glass Ind. Ltd., Osaka, Japan). The cover glasses were placed in culture dishes, and 1.5 ml a-MEM containing 10% FBS and antibiotics were added. The cells were precultured for 3 days. To apply compressive force, cells on coverslips were placed on an appliance that we designed (Fig. 1A). Compressive force was applied continuously by changing the amount of culture medium. PDL cells were subjected to 0.17 (control) or 2.0 g/ cm2 compressive force for 2, 3, or 4 days. To examine the effects of neuropeptides, we added SP (108 M; Peptide Institute, Osaka, Japan) or CGRP (108 M; Peptide Institute) to the medium. Furthermore, we investigated the effects of SP antagonist (107 M, spantide, Peptide Institute) and CGRP antagonist (107 M, calcitonin gene-related peptide fragment 8–37, CGRP8–37; Sigma Chemicals, UK) when the cells were cultured with 108 M SP or 108 M CGRP under the compressive forces.

2.3.

Materials and methods

2.1.

Cell culture

Human PDL cells were isolated from extracted premolars obtained for orthodontic reasons from healthy young donors at Kyushu Dental College Hospital, Kitakyushu, Japan. All of the study followed the guidelines of the Research Ethics Committee of Kyushu Dental College, and informed consent was obtained from all volunteers. The cells were isolated by scraping the ligament tissue from the middle third of the tooth root, mincing the tissue with a scalpel, and digesting it with 2 mg/ml collagenase type IV (Sigma, St. Louis, MO) in phosphatebuffered saline (PBS) supplemented with antibiotics (167 U/ ml penicillin G, 50 mg/ml gentamicin, and 0.25 mg/ml fungizone) for 30 min at 37 8C. The cells were collected by centrifugation and suspended in a-minimal essential medium (aMEM; Gibco, Grand Island, NY) supplemented with antibiotics and containing 10% fetal bovine serum (FBS; Gibco).

Cell morphology

To evaluate the cell condition, the morphology of PDL cells that had been subjected to compressive force or neuropeptides were examined under a phase-contrast microscope (ULWCD 0.30, Olympus Optical, Tokyo, Japan) equipped with an Olympus SC35 type 12 camera (Olympus).

2.4.

Cell count

The effects of compression or neuropeptides on cell proliferation were examined by counting the number of PDL cells. The cells were incubated with 1 ml 0.25% trypsin (Gibco) for 5 min at 37 8C, and the number of PDL cells was counted using Glasstic silde (Hycor Biomedical Inc., CA).

2.5.

2.

Compressive force application

Immunofluorescence staining

To confirm the expression of NK1-R in PDL cells, we used immunofluorescence staining. PDL cells plated on coverslips were washed with PBS, and fixed in 4% paraformaldehyde for 10 min. After washing again with PBS for 5 min, the cells were blocked with 10% goat serum (Nichirei, Tokyo, Japan) for 30 min, washed three times in PBS, and then incubated with anti-NK1-R rabbit IgG antibody (1:1000; Calbiochem, San Diego, CA) for 2 h at 37 8C. After another three washes with PBS, the samples were incubated with Alexa Fluor 488 goat anti-rabbit IgG antibody (1:400; Molecular Probes Inc, Eugene, OR) for 2 h at 37 8C. Actin staining was performed as described by Opas [28] with a few modifications. Cells were washed in cytoskeleton stabilization buffer (0.1 M PIPES, 1 mM EGTA, 4% (w/v) polyethylene glycol 8000 [all Sigma], pH 6.9) three times. After washing, cells were incubated with TRITC-labeled phalloidin (1:40; Molecular Probes) for 45 min at 37 8C. The samples were examined using a fluorescence microscope (Olympus) equipped with a CoolSNAP CCD camera (RS Photometrics, Tucson, AZ).

orthodontic waves 66 (2007) 33–40

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Fig. 1 – (A) Isolated human periodontal ligaments (PDL) cells were seeded in culture dishes and incubated at 37 8C in an atmosphere of 5% CO2. Then, compression forces were applied by changing the amount of medium. The forces produced by the medium were 0.17 g/cm2 (1.5 ml; control) and 2.0 g/m2 (17.6 ml). A total of 10S8 M SP or CGRP was added to the medium. (B) NK1-R mRNA expression in unstimulated PDL cells was confirmed by RT–PCR using primers specific to the NK1-receptor. Molecular products were recognized as a band at 152 bp. (C–F) Double staining for actin (C and E) and NK1-R (D and F) in unstimulated PDL cells. NK1-R localized mainly at the center of the cell (D). The distribution of NK1-R is recognized as punctate immunofluorescent reactions (F). The scale bars represent 10 mm (C and D) and 1 mm (E and F).

2.6.

Semi-quantitative RT–PCR

Total cellular RNA was isolated from each culture using a Total RNA Extraction Miniprep system (Viogene, Sunnyvale, CA), according to the manufacturer’s protocol. Total RNA was extracted from the cells after compression for 2, 3, or 4 days. cDNA was synthesized from 2.0 mg total RNA in 30 ml reaction buffer composed of 500 mM dNTPs, 20 U ribonuclease inhibitor (Promega, Madison, WI), and 200 U Superscript II reverse transcriptase (Invitrogen Life Technology, Carlsbad, CA). Oligonucleotide primer sequences were designed for the reverse transcriptase–polymerase chain reaction (RT–PCR), and their specificities were confirmed using a BLAST-assisted Internet search (National Library of Medicine, Bethesda, MD). The following primers were used for amplification: for RANKL, 50 -TCA GAA GAT GGC ACT CAC TG-30 and 50 -AAC ATC TCC CAC

TGG CTG TA-30 ; for OPG, 50 -AGG CCC TTC AAG GTG TCT TGG TC-30 and 50 -GTG GTG CAA GCT GGA ACC CCA G-30 ; for GAPDH, 50 -TGG TAT CGT GGA AGG ACT CAT G-30 and 50 -TCT CTT CCT CTT GAG CTC TTG C-30 ; for NK1-R I, 50 -TCT TCT TCC TCC TGC CCT ACA TC-30 and 50 -GGT TGG ATC CTC ACC TGT CAT-30 ; and for CGRP receptor (calcitonin receptor-like receptor; CRLR), 50 GTA ATG TTA ACA CCC ACG AGA AAG-30 and 50 -ATC CCC AGC CAA GAA AAT AAT AC-30 . Each cycle consisted of three steps: denaturation at 94.0 8C (RANKL, 60 s; OPG, 40 s; GAPDH, 30 s; NK1-R, 60 s; CRLR, 60 s), annealing (RANKL, 54 8C for 60 s; OPG, 64.0 8C for 40 s; GAPDH, 60.0 8C for 60 s; NK1-R, 62.0 8C for 60 s; CRLR, 55.0 8C for 60 s), and extension at 72.0 8C (RANKL, 60 s; OPG, 40 s; GAPDH, 90 s; NK1-R, 60 s; CRLR, 60 s). The PCR products were electrophoresed in 2% agarose gels, and visualized with ethidium bromide. The data were analyzed using NIH Image.

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2.7.

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Enzyme-linked immunosorbent assay (ELISA)

Detection of soluble RANKL (sRANKL) and OPG were measured by ELISA using the specific antibodies and ELISA amplication system (Invitrogen Life Technology, Carlsbad, CA). PDL cells were cultured in the various conditions as mentioned above, 100 ml of the medium from each sample were pipeted into the wells of 96-well plastic plates and incubated at 4 8C overnight. All samples were assayed in triplicate. After incubation, the plates were washed with PBS-Tween (0.5%, v/v, Tween 20) and the plates were blocked with bovine serum albmin (BSA) in 0.1 M PBS for 30 min at room temperature. Then, the plates were incubated with anti-human polyclonal RANKL antibody (1:300 dilution; Santa Cruz Biotechnology, Santa Cruz, CA) or anti-human polyclonal OPG antibody (1:200 dilution; CHEMICON International, Temecula, CA) for 2 h at room temperature. And after washing, the detecting biotinylated anti-rabbit IgG (1:3000 dilution; Zymed Labolatories, South San Francisco, CA) was added and the plates were incubated for 30 min at room temperature. Next, alkalinephosphatase-avidin (1:1000 dilution; Zymed Laboratories) was added and incubated for 30 min at room temperature. After further washes, the alkaline phosphatase activity was detected with ELISA amplication system. The optical density was measured at 495 nm using a micro plate reader (Bio-Rad, Herts, UK). Protein assay was performed using bicinchoninic acid (BCA) assay method (Pierce, Rockford, IL).

2.8.

Statistical analysis

One-way ANOVA was used to analyze cell damage, followed by individual post hoc comparisons (Scheffe´).

3.

Results

To examine the expression of NK1-R in PDL cells, RT–PCR was performed using NK1-R-specific primers. NK1-R expression was detected in PDL cells at the molecular weight 152 bp (Fig. 1B). To confirm our findings regarding NK1-R expression in PDL cells, we performed immunocytochemistry using an antibody against NK1-R, which showed that many punctate immunopositive sites for NK1-R were distributed in unstimulated PDL cells stained by TRIRC-phalloidin (Fig. 1C–F). The distribution of NK1-Rs in compressed PDL cells were similar as that in unstimulated cells. Regarding with CGRP receptors, CRLR expression was hardly detected in PDL cells by RT-PCR. We used 2.0 g/cm2 compressive force and 108 M SP or 8 10 M CGRP. This compression and the concentrations of neuropeptides were determined based on the results of preliminary experiments and previous studies [11,29]. The in vitro application of 4 days of compression altered the morphology of PDL cells (Fig. 2A and B). With compression, atrophic cells were observed. The morphology of PDL cells with compression and SP was similar to those with only compression (Fig. 2C). With the addition of CGRP, cell morphology was similar to cells with SP (data not shown). Although compression altered the morphology of PDL cells, the number of cells after compression, with or without SP, was not significantly different from the control (Fig. 2D). To evaluate differences in the expression of bone-related genes, the expression of OPG and RANKL mRNA was measured by RT–PCR (Fig. 3A and B). Very little RANKL mRNA expression was detected in unstimulated cells. Compression increased RANKL mRNA expression in a time-dependent manner, reaching a maximum at day 4. With the addition of SP or

Fig. 2 – Cell condition assessed by phase-contrast images of PDL cells, and the number of cells after compressive force for 4 days. The cells were cultured (A) without additional medium (control), (B) with 2.0 g/cm2 compressive force, or (C) with 10S8 M SP and 2.0 g/cm2 compressive force. Atrophic cells appear bright (arrows). Scale bars = 20 mm. (D) Number of cells without additional medium (control), with 2.0 g/cm2 compressive force (load), and with 10S8 M SP and compressive force (SP + load). Data are presented as means W S.D.

orthodontic waves 66 (2007) 33–40

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Fig. 3 – (A) RANKL, OPG, and GAPDH mRNA expression in PDL cells under compression (load) and with or without 10S8 M SP or 10S8 M CGRP. Stimulation was applied for 2–4 days. Total RNA was analyzed using RT–PCR. MW indicates molecular weight markers. Molecular products for RANKL and OPG were recognized as bands at 879 bp and 647 bp, respectively. (B) RANKL and OPG mRNA expression relative to GAPDH expression was analyzed using NIH Image. The band intensities of OPG expression at day 2 in the control, and RANKL expression at day 4 with compression force (maximum values) were assigned values of 1. (C) To confirm the effect of neuropeptides on PDL cells, PDL cells were cultured in the presence of 10S8 M SP, 10S8 M SP + 10S7 M spantide (SP antagonist), 10S8 M CGRP, or 10S8 M CGRP + 10S7 M CGRP antagonist (CGRP a) with compression for 4 days. MW indicates molecular weight markers. (D) OPG mRNA expression relative to GAPDH expression was analyzed using NIH Image. The band intensity of OPG expression at day 2 in the control was assigned values of 1.

CGRP, weak expression of RANKL mRNA was seen. When SP or CGRP was added to compressed cells, RANKL mRNA expression was higher than in uncompressed cells. However, RANKL mRNA expression in compressed cells with SP or CGRP was lower than in compressed cells without SP or CGRP. Thus, SP or

CGRP impaired the increase in RANKL mRNA expression induced by compression. The highest OPG mRNA expression level was detected in unstimulated PDL cells. Compressed cells expressed less OPG mRNA than control cells. When cells were incubated in the

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presence of SP, OPG expression was lower than in control cells. Furthermore, the addition of SP decreased OPG mRNA expression in compressed cells. The addition of SP inhibited OPG mRNA expression synergistically with compression (control: 0.70; load: 0.43; SP and load: 0.21; at day 4 relative to controls at day 2). We also examined the effects of CGRP on PDL cells. Like SP, there was less OPG mRNA in the CGRP stimulated cells than in control cells. The addition of CGRP also inhibited OPG mRNA expression synergistically with compression. To confirm that SP or CGRP affects the function of PDL cells through their receptors, we investigated the effects of SP or CGRP antagonists on OPG and RANKL expressions in PDL cells that were cultured with 108 M SP or 108 M CGRP under the compression forces for 4 days. SP or CGRP antagonists increased the expression of OPG mRNA inhibited by SP or CGRP (Fig. 3C and D) and inhibited the neuropeptide-induced RANKL expressions, which were hardly detected by RT-PCR (data not shown). ELISA assays were used to evaluate the effect of neuropeptides on sRANKL and OPG production from PDL cells (Fig. 4). The production of sRANKL and OPG showed same tendency as RT-PCR assay. Without compression or neuropeptides no production of sRANKL was detected. Compression induced sRANKL production, and significantly increased sRANKL production stimulated by 108 M CGRP. sRANKL production was also increased by the combination of SP and compression

Fig. 4 – Enzyme-linked immunosorbent assay (ELISA) for the effect of neuropeptides and load on the production of sRANKL and OPG from PDL cells at 4 days. (A) The production of sRANKL under compressinon was assigned a value of 1. (B) The production of OPG in controls was assigned a value of 1. Each value represents mean W S.E.M. Significant difference (*P < 0.05) between groups (n = 8).

compared with SP only, but no significant difference between them. However, sRANKL production in compressed cells with SP or CGRP was lower than in compressed cells without 108 M SP or 108 M CGRP (Fig. 4A). Compression decreased OPG production. The addition of SP or CGRP also significantly inhibited OPG production synergistically with compression (Fig. 4B).

4.

Discussion

We demonstrated that compressed PDL cells down-regulate OPG and up-regulate RANKL expression. The neuropeptides SP and CGRP synergistically decreased OPG mRNA expression in compressed PDL cells, whereas they suppressed the RANKL expression induced by compression. These results suggest that SP and CGRP may modulate bone remodeling by PDL cells during orthodontic tooth movement. NK1-Rs are found not only in neuronal and immune cells but also in oral tissue, including rat PDL cells [30–32]. Here we clarified that human PDL cells are targets for neuropeptides by examining the expression of NK1-R. The CGRP receptor is found in developing rat periodontal ligaments [33]. However, it is unclear if the CGRP receptor is localized in PDL cells. We examined the expression of CGRP receptors, however, no obvious expression was observed. Further studies are needed to determine if CGRP receptors are localized in human PDL cells. Various methods have been used to apply compression or extension in vitro. We designed a new method for applying compression by increasing the volume of medium to create hydraulic pressure. Using this method, uniform pressure could be applied to the cells, and sufficient nutrition was provided. In preliminary experiments, we varied the amount of pressure from 0.17 to 7.0 g/cm2, and found that 2.0 g/cm2 was suitable. A 0.17 g/cm2 compressive force was too weak to induce physiological changes in PDL cells. A compression force of 7.0 g/cm2 was so strong that it decreased the number of cells. In our preliminary experiments we found the compressive forces over 4 days increased atrophied cells. Compressive force damaged PDL cells, and caused them to atrophy, which is consistent with a previous study [34]. Therefore, we used 2–4 days compression forces in the present study. As it is not known whether sensory neuropeptides affect PDL cells, we first examined the effects of neuropeptides on the proliferation of PDL cells. Previous studies have shown that SP stimulates cell proliferation [29,35,36]. However, in this study, SP and CGRP did not stimulate the proliferation of PDL cells, although they affected OPG and RANKL mRNA expression. These findings suggest that human PDL fibroblastic cells are less affected by sensory neuropeptides than fibroblastic cells in lung or synovial membranes [29,36]. Here, compression increased RANKL mRNA expression, which is consistent with a previous study [37]. Some studies have indicated that SP up-regulates the production and expression of RANKL mRNA [29,38]. We also found an increase in RANKL mRNA expression with the addition of SP. On the contrary, CGRP may not influence RANKL expression [20]. In our study, the addition of CGRP increased RANKL mRNA

orthodontic waves 66 (2007) 33–40

expression compared to the control. Although SP and CGRP increased RANKL mRNA expression, the amount of RANKL mRNA did not increase as much as when compression was applied. Furthermore, SP and CGRP with compression decreased RANKL mRNA expression. These findings suggest that the effects of sensory neuropeptides on RANKL mRNA expression are less than those of compressive forces. OPG, a member of the TNF receptor family, is a soluble decoy receptor for RANKL, and an inhibitor of osteoclastgenesis [39]. Generally, osteoclastogenesis is thought to require both RANKL up-regulation and OPG down-regulation. Previous studies indicated that mechanical stress (tensile force) upregulates OPG mRNA expression in a force-dependent manner [40], whereas OPG expression remains constant under compressive forces [37]. In this study, both compressive forces and neuropeptides decreased OPG mRNA expression. Our results are consistent with previous reports that SP and CGRP both decrease OPG mRNA expression [29,41]. In contrast to its effects on RANKL mRNA expression, SP inhibited OPG mRNA expression synergistically with the application of compression forces. Taken together, these results suggest that sensory neuropeptides mainly affect the expression of OPG mRNA and have lesser effects on RANKL mRNA expression in compressed PDL cells. In conclusion, SP and CGRP increased the expression of RANKL mRNA in PDL cells, whereas in compressed PDL cells SP and CGRP decreased OPG and RANKL mRNA expression. Therefore, the neuropeptides SP and CGRP may be involved in bone remodeling primarily through OPG mRNA expression in PDL cells during orthodontic tooth movement.

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