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Parathyroid-hormone-related Protein Induces Expression of Receptor Activator of NF-B Ligand in Human Periodontal Ligament Cells via a cAMP/Protein Kinase A-independent Pathway

¹ Department of Physiological Science and Molecular Biology and
² Department of Oral Growth and Development, Fukuoka Dental College, Tamura 2-15-11, Sawara-ku, Fukuoka 814-0193, Japan;

^* corresponding author, ejimi{at}college.fdcnet.ac.jp

	ABSTRACT

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Periodontal ligament (PDL) cells play important roles in root resorption of human deciduous teeth by odontoclasts (osteoclast-like cells). However, it is unclear how PDL cells regulate osteoclastogenesis. We examined the effects of PTHrP, TGF-ß, and EGF, which are all secreted by the tooth germ, on tartrate-resistant acid-phosphatase-positive (TRAP⁺) cell formation using co-cultures of human PDL cells and mouse spleen cells. Only PTHrP promoted TRAP⁺ cell formation in co-cultures. PTHrP induced receptor activator of NF-B ligand (RANKL) mRNA expression and slightly reduced osteoprotegerin (OPG) expression in PDL cells. The cAMP/PKA inhibitors Rp-cAMP, H89, and PKI did not affect PTHrP-induced TRAP⁺ cell formation. The PKC inhibitor, Ro-32-0432, suppressed RANKL expression in PDL cells and PTHrP-induced TRAP⁺ cell formation. However, this inhibitor directly modulated the number of osteoclast precursors. Thus, PTHrP induces osteoclastogenesis by increasing the relative expression level of RANKL vs. OPG in PDL cells via a cAMP/PKA-independent pathway. Abbreviations: PTHrP, parathyroid-hormone-related protein; TGF-ß, transforming growth factor-ß; EGF, epidermal growth factor; RANKL, receptor activator of NF-B ligand; OPG, osteoprotegerin; PDL, periodontal ligament; TRAP, tartrate-resistant acid phosphatase; PKA, protein kinase A; PKC, protein kinase C; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; cAMP, cyclic Adenosine 3'5'-Monophosphate.

KEY WORDS: PTHrP • periodontal ligament • osteoclast • PKA • PKC

	INTRODUCTION

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Physiological root resorption during the shedding of human deciduous teeth leads to eruption of permanent teeth and the formation of functional occlusion (Ten Cate and Anderson, 1986). In vivo and in vitro studies have demonstrated that odontoclasts, tartrate-resistant acid-phosphatase-positive (TRAP⁺) multinucleated cells (MNCs), play a central role in the root resorption of deciduous teeth, and form an eruption pathway for permanent teeth (Boyde and Lester, 1967; Sasaki et al., 1990; Sahara et al., 1996). It has been thought that there is little difference between odontoclasts and osteoclasts in their structural and histochemical properties, such as expression of TRAP and receptor activator of NF-B (RANK) (Oshiro et al., 2001). Despite these similarities to osteoclasts, the mechanisms regulating odontoclast formation and root-resorbing activity are poorly understood.

Since the discovery of the RANK ligand (RANKL) and its decoy receptor osteoprotegerin (OPG), it has been believed that the balance of these two factors is critical for regulating osteoclast differentiation and function (Wagner and Karsenty, 2001; Theill et al., 2002). Cells of the periodontal ligament (PDL), located between the alveolar bone and the roots of teeth, secrete OPG and prevent resorption of these hard tissues by inhibiting osteoclast formation (Shimizu et al., 1996; Kanzaki et al., 2001). Recently, we demonstrated that human PDL cells derived from roots of resorbing deciduous teeth expressed increased levels of RANKL and decreased levels of OPG, while PDL cells isolated from roots of either stable/non-resorbing deciduous or permanent teeth expressed OPG but not RANKL (Fukushima et al., 2003). Analysis of these data demonstrates the importance of RANKL/OPG signaling pathways for physiological root resorption.

The tooth germ, including dental follicle and the epithelial stellate reticulum, also contributes to physiological root resorption of deciduous teeth during eruption of permanent teeth, by releasing several growth factors and cytokines, such as PTHrP, TGF-ß, and EGF (Wise et al., 2002). Indeed, removal of the dental follicle from premolars prior to the onset of eruption has been shown to prevent tooth eruption (Marks and Cahill, 1984), suggesting that factors released from the tooth germ contribute to this process. However, it is unclear to what extent these growth factors and cytokines secreted by the tooth germ are involved in regulation of RANKL/OPG expression in PDL cells.

In the present study, we examined the effects of PTHrP, TGF-ß, and EGF, which are all secreted by the tooth germ, on tartrate-resistant acid-phosphatase-positive (TRAP⁺) cell formation, using co-cultures of mouse spleen cells and human PDL cells derived from stable/non-resorbing deciduous teeth.

	MATERIALS & METHODS

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All procedures involving animals were approved by the Council on Animal Care at Fukuoka Dental College. The protocol for the present experiments was reviewed and approved by the Fukuoka Dental College Research Ethics Committee, Fukuoka, Japan, and informed consent was obtained from all volunteers.

Chemicals
Recombinant human transforming growth factor-ß (TGF-ß), human epidermal growth factor (EGF), and human PTHrP (1-34) were obtained from the R&D Co. (Minneapolis, MN, USA) and Bachem AG (Torrance, CA, USA), respectively. Recombinant human RANKL, OPG, soluble RANK, and macrophage colony-stimulating factor (M-CSF) were purchased from PeproTech Inc. (Rocky Hill, NJ, USA). All inhibitors were purchased from Calbiochem-Novabiochem (La Jolla, CA, USA). All other chemicals were obtained from Sigma Chemical Co. (St. Louis, MO, USA).

Preparation of PDL Cells
PDL cells were obtained from healthy human non-resorbing deciduous teeth of five donors (from 6 to 8 yrs old) according to an IRB-approved protocol and with informed consent as described previously (Fukushima et al., 2003). Only the middle of each root was used, to exclude contamination by gingivae and dental pulp. Cells that grew from the extracts and underwent from 4 to 8 passages were used in these experiments.

TRAP⁺ Cell Formation and Pit Formation Assay
Mouse spleen cells (1 x 10⁶ cells/well) from four-week-old male ddY mice and PDL cells (1 x 10⁵ cells/well) were co-cultured with or without PTHrP (10^–8 M) in the presence of 10^–7 M dexamethasone (Dex) in 12-well plates coated with collagen gel for 10 days. After 10 days, cells were detached with collagenase and dispase in PBS. Cells were collected and re-suspended into 12-well plates. After 2 hrs, the cultures were fixed with 3.7% formaldehyde and stained for TRAP, a marker enzyme of osteoclasts. TRAP⁺ cells were counted. All inhibitors were present in the cultures prior to PTHrP or RANKL addition. When old media were replaced with fresh media, inhibitors also were added to the culture prior to PTHrP. Data shown are the number of TRAP⁺ cells per culture well (values are mean ± SEM, n = 3 culture wells). Similar results were obtained in 3 independent experiments with PDL cells from three patients. Pit formation by TRAP⁺ cells was performed as described previously (Suda et al., 1997).

RT-PCR
For semi-quantitative RT-PCR, total RNA from PDL cells prepared with Trizol (Invitrogen, Carlsbad, CA, USA) was amplified by Superscript II and Taq polymerase (Invitrogen). Primer sequences of human RANKL, OPG, and GAPDH were described previously (Fukushima et al., 2003). The PCR program was as follows: 35 cycles for RANKL, 30 cycles for OPG, and 22 cycles for GAPDH at 94°C for 40 sec, 60°C for 40 sec, and 72°C for 1 min. Fluorescence of each PCR product was detected by means of an image analyzer (Fluoro Image Analyzer FLA-2000F, Fuji Film, Tokyo, Japan). Signals of RANKL, OPG, and GAPDH mRNA were quantitated and normalized with the respective GAPDH mRNA expression levels, for calculation of relative intensity, with the use of an NIH image analyzer.

PKA Assay
Human PDL cells were treated with PTHrP (10^–8 M) for 20 min, and cells then underwent lysis in a TNT buffer containing protease inhibitors and phosphatase inhibitors. A 20-µg quantity of protein was used for PKA reaction with a PepTag assay kit (ProMega, Madison, WI, USA), following the manufacturer’s protocol.

Flow Cytometry
Mouse bone marrow cells (BMCs) were pre-treated with Ro-32-0432, followed by incubation with M-CSF (100 ng/mL). After 3 days, cells were stained with FITC-conjugated anti-c-fms monoclonal antibody (AFS98, eBioscience, San Diego, CA, USA) for 30 min. Cells were washed and analyzed with a FACScan flow cytometer (Becton Dickinson).

Data Analysis
Statistical differences were analyzed by one-way ANOVA, and P values less than 0.05 were considered to be significant.

	RESULTS

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Effects of PTHrP on TRAP⁺ Cell Formation in Co-cultures of Human PDL Cells and Mouse Spleen Cells
We first examined the effects of PTHrP (10^–8 M), TGF-ß (10 ng/mL), or EGF (10 ng/mL) on TRAP⁺ cell formation in co-cultures of human PDL cells and mouse spleen cells in the presence of dexamethasone (Dex) (10^–7 M) for 10 days. The human PDL cells expressed all receptors for these factors (data not shown). Of these factors, only PTHrP promoted TRAP⁺ cell formation in our co-cultures (Fig. 1A). PTHrP strikingly increased the number of TRAP⁺ cells at a concentration of 10^–8 M (Figs. 1B, 1C). TRAP⁺ cells induced by PTHrP showed characteristics typical of osteoclasts, such as the ability to form resorption pits on dentin slices (Fig. 1C, lower panels).

View larger version (26K): [in this window] [in a new window]   Figure 1. Effects of PTHrP on TRAP+ cell formation in co-cultures of human PDL cells with mouse spleen cells. (A) Mouse spleen cells (1 x 105 cells/well) were co-cultured with PDL cells (1 x 105 cells/well) in the presence or absence of PTHrP (10–8 M), TGF-ß (10 ng/mL), or EGF (10 ng/mL) in the presence of Dex (10–7 M) for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in three independent experiments. *P < 0.01 vs. control cultures. (B) Mouse spleen cells (1 x 105 cells/well) were co-cultured with PDL cells (1 x 105 cells/well) in various concentrations of PTHrP together with Dex for 10 days. TRAP+ cells were counted and expressed as the number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in three independent experiments. *P < 0.01 vs. control cultures. (C) Microscopic view of TRAP+ cells. Resorption areas were stained with Mayer’s hematoxylin (lower panels). Bar = 100 µm. (D) Human PDL cells were treated with PTHrP (10–8 M) in the presence of Dex for the indicated time. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (E) Co-cultures were pre-treated with or without either OPG (100 ng/mL) or sRANK (100 ng/mL) for 30 min, followed by incubation with PTHrP (10–8 M) in the presence of Dex for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures.

Inhibition of the cAMP/PKA Signaling Pathway Does Not Inhibit PTHrP-induced TRAP⁺ Cell Formation in Co-cultures of Human PDL Cells and Mouse Spleen Cells
Previous work has shown that PTHrP induced osteoclastogenesis in mouse bone marrow cultures via a cAMP mechanism (Akatsu et al., 1989a). Co-cultures were pre-treated with cell-permeable Rp-cAMP (100 µM), a specific inhibitor of cAMP, and then cultured with PTHrP for 10 days. Unexpectedly, Rp-cAMP did not affect TRAP⁺ cell formation induced by PTHrP in our co-cultures (Figs. 2A, 2B). Next, we pre-treated co-cultures with PKA inhibitors H89 (1 µM) or cell-permeable PKI (1 µM), and these inhibitors also failed to suppress PTHrP-induced TRAP⁺ cell formation (Fig. 2A). In contrast, the addition of PKI greatly inhibited TRAP⁺ cell formation induced by PGE₂, which is known to induce osteoclastogenesis via a cAMP/PKA signaling pathway (Fig. 2A) (Akatsu et al., 1989b). Consistent with TRAP⁺ cell formation, PKI did not affect PTHrP-induced RANKL expression in PDL cells (Fig. 2C), although the PTHrP-induced PKA activity in PDL cells decreased to 80% after pre-treatment with PKI (1, 10 µM) (Figs. 2D, 2E). Pre-treatment with H89 (1 µM) or PKI (1 µM) did not affect PTHrP-induced osteoclastogenesis in a co-culture of mouse calvaria cells with mouse spleen cells, indicating that this phenomenon is not specific for human PDL cells (Figs. 2F, 2G). These results strongly suggest that PTHrP-induced TRAP⁺ cell formation is mediated by a cAMP/PKA-independent mechanism.

View larger version (42K): [in this window] [in a new window]   Figure 2. Inhibition of the cAMP/PKA signaling pathway did not inhibit PTHrP-induced TRAP+ cell formation in co-cultures. (A) Co-cultures were pre-treated with or without Rp-cAMP (100 µM), H89 (1 µM), or PKI (1 µM) for 30 min, followed by incubation with either PTHrP (10–8 M) or PGE2 (10–8 M), together with Dex (10–7 M), for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PGE2-treated cultures. (B) Microscopic view of TRAP+ cells. Bar = 100 µm. (C) Human PDL cells were pre-treated with PKI (1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (D) Human PDL cells were pre-treated with or without PKI (0.1, 1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) or PGE2 (10–8 M) for 20 min together with Dex. Total cell lysates were used for PKA assay, with the PepTag PKA assay kit. Similar results were obtained in 3 independent experiments. (E) PKA activity was measured by densitometry. Each column indicates the relative value of phosphorylation by PKA vs. the intensity of the phosphorylation signal without PTHrP. *P < 0.01 vs. PTHrP or PGE2-treated cultures. (F) Co-cultures of mouse bone marrow cells (1 x 105 cells/well) and mouse calvaria cells (1 x 105 cells/well) were pre-treated with or without H89 (1 µM) or PKI (1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) for 5 days. TRAP+ multinucleated cells (MNCs) were counted. Data shown are the number of TRAP+ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. (G) Microscopic view of TRAP+ cells and MNCs. Bar = 100 µM.

View larger version (24K): [in this window] [in a new window]   Figure 3. PKC pathway is partially involved in PTHrP-induced TRAP+ cell formation in co-cultures. (A) Co-cultures of mouse spleen cells and human PDL cells were pre-treated with or without PD98059 (10 µM) (ERK inhibitor), NS-398 (10 µM) (COX2 inhibitor), SB203580 (10 µM) (p38 MAPK inhibitor), and Ro-32-0432 (1 µM) (PKC inhibitors) for 30 min, followed by incubation with PTHrP (10–8 M) together with Dex for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures. (B) Human PDL cells were pre-treated with or without SB203580 (10–7, 10–6 M) for 30 min, followed by incubation with PTHrP (10–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (C) We cultured mouse bone marrow cells (BMCs) with M-CSF for 3 days to prepare bone marrow macrophages (BMMs). BMMs were pre-treated with or without SB03580 (10–7, 10–6 M) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 3 days. Data shown are number of TRAP+ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. RANKL-treated cultures. (D) Co-cultures of mouse spleen cells and human PDL cells were pre-treated with or without Ro-32-0432 (0.1 and 1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) together with Dex for 10 days. TRAP+ cells were counted and expressed as number of TRAP+ cells per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. PTHrP-treated cultures. (E) Human PDL cells were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with PTHrP (10–8 M) for 72 hrs. Total RNA was isolated from PDL cells, and expression levels of RANKL, OPG, and GAPDH mRNA were measured by RT-PCR analysis. Numbers below the gels represent n-fold increase in intensity of RANKL or OPG relative to corresponding GAPDH mRNA signals. Similar results were obtained in 3 independent experiments. (F) We cultured mouse BMCs with M-CSF for 3 days to induce BMMs. BMMs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 3 days. Mouse BMCs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with RANKL (100 ng/mL) in the presence of M-CSF (100 ng/mL) for 5 days. Data shown are number of TRAP+ MNCs per culture well (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. RANKL-treated cultures. (G) Mouse BMCs were pre-treated with or without Ro-32-0432 (0.1, 1 µM) for 30 min, followed by incubation with M-CSF (100 ng/mL) for 3 days. After 3 days, cells were stained with FITC-conjugated anti-c-fms antibody, and c-fms+ cells were counted by flow cytometry (values are mean ± SEM, n = 3). Similar results were obtained in 3 independent experiments. *P < 0.01 vs. M-CSF-treated cultures.

	DISCUSSION

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Among factors released from the tooth germ, the significance of the PTHrP signaling pathway molecules for tooth eruption has been demonstrated in vivo, since PTHrP gene-targeting in mice resulted in failure of tooth eruption (Philbrick et al., 1998; Kitahara et al., 2002). It was reported that PTHrP induced osteoclastogenesis in mouse bone marrow cultures via a mechanism involving cAMP/PKA and calcium ions, and that PTHrP-induced cAMP accumulation in mouse bone marrow cells was dose-dependent (Akatsu et al., 1989a). Since then, other studies have suggested that PTH/PTHrP-induced biological effects in osteoblastic cells are mediated by both cAMP/PKA-dependent and -independent pathways (Tetradis et al., 1997; Carpio et al., 2001; Kawane et al., 2003). We show here that pre-treatment with cAMP/PKA inhibitors does not affect TRAP⁺ cell formation in co-cultures, even though these inhibitors suppressed up to 80% of the PTHrP-induced PKA activity. These inhibitors also failed to suppress osteoclastogenesis induced by PTHrP in co-cultures of mouse calvaria cells with bone marrow cells.

It is possible that p38 MAP kinase and PKC pathways are involved in PTHrP-induced TRAP⁺ cell formation in co-cultures. However, consistent with a previous report (Li et al., 2002), SB203580 did not affect RANKL induction by PTHrP in human PDL cells, and inhibited RANKL-induced osteoclastogenesis from mouse BMMs. Therefore, we can exclude involvement of the p38 pathway on PTHrP-induced RANKL expression in human PDL cells. In contrast, consistent with a previous report (Takami et al., 2000), Ro-32-0432 significantly suppressed the expression level of RANKL mRNA induced by PTHrP in human PDL cells. We also found that the number of spleen cells was reduced during the co-culture period (data not shown), suggesting that Ro-32-0432 has a direct effect on osteoclast precursors. Consistent with this observation, Ro-32-0432 decreased the number of c-fms⁺ cells that differentiated from BMCs induced by M-CSF. Although, at present, we cannot precisely determine the contribution of the PKC pathway to TRAP⁺ cell formation induced by PTHrP, PKC-dependent RANKL induction is likely to be involved in the PTHrP-induced TRAP⁺ cell formation in our co-cultures. In addition, the PKC pathway might also directly modulate the number of osteoclast precursors (Fig. 4).

View larger version (32K): [in this window] [in a new window]   Figure 4. Schematic representation of PTHrP-induced osteoclast formation. PTHrP induces RANKL expression in PDL cells by a mechanism independent of the cAMP/PKA signaling pathway. PKC contributes to induction of RANKL mRNA induced by PTHrP in PDL cells as well as regulation of osteoclast precursor cell number. PGE2 induces RANKL expression in PDL cells via the cAMP/PKA signaling pathway.

	ACKNOWLEDGMENTS

 
We thank Dr. Andreas Carl for English editing. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology, Japan (No. 14571784) and by a Frontier Research Grant.

Received August 24, 2004; Last revision December 20, 2004; Accepted January 4, 2005

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