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Mechanical Stress Induces Osteopontin via ATP/P2Y1 in Periodontal Cells
S. Wongkhantee,
T. Yongchaitrakul and
P. Pavasant*
Department of Anatomy and Graduate School of Oral Biology, Faculty of Dentistry, Chulalongkorn University, Henri Dunant Road, Pathumwan, Bangkok 10330, Thailand
Correspondence: * corresponding author, prasit215{at}gmail.com
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ABSTRACT
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Our previous study showed that mechanical stress induced the expression of osteopontin (OPN) in human periodontal ligament (HPDL) cells through the Rho kinase pathway. The increase of OPN expression via Rho kinase has been demonstrated to be triggered by nucleotide. Therefore, we hypothesized that nucleotides, particularly adenosine triphosphate (ATP), participated in the stress-induced OPN expression in HPDL cells. In the present study, the roles of ATP and P2Y1 purinoceptor were examined. Reverse-transcription polymerase chain-reaction and Western blot analysis revealed that the stress-induced ATP exerted its stimulatory effect on OPN expression. The inductive effect was attenuated by apyrase and completely inhibited by the Rho kinase inhibitor, as well as by the P2Y1 antagonist. We here propose that stress induces release of ATP, which in turn mediates Rho kinase activation through the P2Y1 receptor, resulting in the up-regulation of OPN. Stress-induced ATP could play a significant role in alveolar bone resorption.
Key Words: ATP • P2Y • osteopontin • stress
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INTRODUCTION
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Human periodontal ligament (HPDL) cells are responsible for the mechanical stress transduced from the tooth, which is significant during periodontal tissue remodeling and repair (Lekic and McCulloch, 1996). Excessive mechanical stress can induce bone loss through the elevation of inflammatory cytokines (Ren et al., 2002; Yamaguchi and Kasai, 2005) and imbalance of the receptor activator of nuclear kappa B ligand (RANKL) and osteoprotegerin (OPG) (Kanzaki et al., 2002; Yamaguchi et al., 2006). It has been demonstrated that mechanical stress also induces the expression of osteopontin (OPN) (Wongkhantee et al., 2007).
The molecular mechanism of OPN induction by mechanical stress in HPDL cells is still unclear. Several reports demonstrated an increase of prostaglandin E2 (PGE2) after stress application in both osteoblasts and HPDL cells (Kanzaki et al., 2002; Rawlinson et al., 2000; Kapur et al., 2003). In addition, activation of mitogen-activated protein kinase (MAPK) by stress has been reported (You et al., 2001). However, using inhibitors, we have indicated that Rho kinase, but neither PGE2 nor MAPK, was involved in the stress-induced OPN in HPDL cells (Wongkhantee et al., 2007).
In the present study, the molecules involved in the stimulation of OPN expression were further investigated. The involvement of Rho kinase in the regulation of OPN expression has been reported in smooth-muscle cells (Chaulet et al., 2001; Kawamura et al., 2004). Induction of OPN was observed when smooth-muscle cells were activated with nucleotides, or when high glucose levels occurred via Rho kinase, suggesting the role of nucleotides in OPN expression. Among the nucleotides, adenosine triphosphate (ATP) has been recognized as an important and ubiquitous intracellular and extracellular messenger in various kinds of tissues (Brambilla and Abbracchio, 2001; Burnstock and Knight, 2004; Smith and Scott, 2006). Intracellular ATP is converted into cAMP by adenylyl cyclase and acts as a major signaling molecule responsible for several biological responses, while extracellular ATP mediates its action through the family of P2 purinoceptors. The P2 purinoceptor is classified into 2 main subfamilies: P2Y, a G-protein coupled receptor; and P2X, an ion channel receptor. At least 8 subtypes of P2Y and 7 subtypes of P2X have been identified (Schwiebert, 2000).
It has been demonstrated that mechanical stress induced a rise in intracellular cAMP and the release of ATP in several cell types, including osteoblasts (Harell et al., 1977; Nakano et al., 1997; Yamamoto et al., 2000; Romanello et al., 2001; Furuya et al., 2005). The released ATP works as an autocrine and paracrine mediator, and plays a role in mechano-transduction. In addition, ATP has a potent stimulatory action on IL-6 secretion, but an inhibitory action on OPG expression (Ihara et al., 2005). ATP also stimulates human osteoclast activity via the up-regulation of RANKL (Buckley et al., 2002). This evidence emphasizes the important role of ATP as one of the regulators of bone homeostasis.
A recent study reported that ATP caused growth arrest in HPDL cells, suggesting the influence of ATP in periodontal tissue regeneration (Kawase et al., 2007). In the present study, we found that HPDL cells expressed P2Y receptors, leading to the hypothesis that these receptors may be significant for the responses of cells to mechanical stress. We thus hypothesized that the up-regulation of OPN expression induced by mechanical stress was caused by the release of ATP, which acted through P2 receptors in HPDL cells.
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MATERIALS & METHODS
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Cell Culture
HPDL cells were obtained from healthy third molars extracted for orthodontic reasons and prepared as previously described (Pattamapun et al., 2003). The protocol was approved by The Ethics Committee, Faculty of Dentistry, Chulalongkorn University, and informed consent was obtained from each person. Briefly, teeth were rinsed with sterile phosphate-buffered saline, and the PDL was removed from the middle third of the root. The explants were harvested on 60-mm culture dishes and grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (Hyclone, Logan, UT, USA), supplemented with 10% fetal calf serum (Hyclone), 2 mM L-glutamine (Gibco BRL, Carlsbad, CA, USA), 100 units/mL penicillin, 100 µg/mL streptomycin (Gibco BRL), and 5 µg/mL amphotericin B (Gibco BRL) in a humidified atmosphere of 95% air, 5% CO2, at 37°C. Cells from the third to the fifth passages were used. All experiments were performed in triplicate with cells prepared from three different donors.
Application of Mechanical Stress
The method for mechanical stress application was modified from Kanzaki et al.(2002). Briefly, cells were seeded in six-well plates at a density of 200,000 cells/well for 16 hrs. A plastic cylinder containing metal coins was placed over the culture to generate compressive forces ranging from 0 to 2 g/cm2.
For inhibitory experiments, each inhibitor was added to the medium 30 min prior to the experiment. The inhibitors used included 15 µM suramin, 0.18 µM NF449, 1 unit/mL apyrase, 0.5–5 µM MRS2179 (all from Sigma-Aldrich Chemical, St. Louis, MO, USA), and 1.25 nM Rhokinase inhibitor (Calbiochem, EMD Biosciences, San Diego, CA, USA).
Application of ATP
Exogenous ATP (0.1, 1, 10 µM) (Sigma-Aldrich) was applied to HPDL cell culture for 2 hrs before total RNA extraction and for 24 hrs before protein extraction. RNA extraction and semi-quantitative transcription polymerase chain-reaction assay (RT-PCR) were performed. Total cellular RNA was extracted (Tri-reagent, Molecular Research Center, Cincinnati, OH, USA) according to the manufacturer’s instructions. One microgram of each RNA sample was converted to cDNA by the use of an avian myeloblastosis virus (AMV) for 1.5 hrs at 42°C. After RT, PCR was performed. The primers were prepared following the reported sequences from GenBank. The oligonucleotide sequences of the primers were: GAPDH (NM002046.3), forward 5'-TGAAGGTCGGAGTCAACGGAT-3', reverse 5'-TCACACCCATGACGAACATGG-3'; OPN (NM000582.2), forward 5'-AGTACCCTGATGCTACAGACG-3', reverse 5'-CAACCAGCATATCTTCATGGC-3'; P2Y1 (NM002563.2), forward 5'-CGGTCCGGGTTCGTCC-3', reverse 5'-CGGACCCCGGTACCT-3'; and P2Y2 (NM002564.2), forward 5'-CTAAAGCCAGCCTACGGGAC-3', reverse 5'-TCCTATCCTCTGCATGTC-3'.
The PCR was performed with Taq polymerase (Qiagen, Hilden, Germany) and a PCR volume of 25 µL. The amplification profile for OPN was 1 cycle at 94°C for 1 min, 30 cycles at 94°C for 1 min, hybridization at 60°C for 1 min,, and extension at 72°C for 2 min, followed by 1 extension cycle at 72°C for 10 min. The same profile was also used for P2Y1, P2Y2 (35 cycles), and GAPDH (22 cycles). The PCR was performed in a DNA thermal cycler (Biometra, Göttingen, Germany). The amplified DNA was subjected to electrophoresis on a 2% agarose gel and visualized by ethidium bromide fluorostaining. The relative intensities of the bands were measured by image analysis.
Protein Extraction and Western Blot Analysis
Protein was extracted with a radioimmunoprecipitation assay (RIPA). Protein concentrations were measured by means of a BCA protein assay kit (Pierce, Rockford, IL, USA). Equal amounts of protein samples were subjected to electrophoresis on a 10% SDS-polyacrylamide gel and subsequently transferred onto nitrocellulose membrane. For the reduction of non-specific binding, the membrane was incubated in 5% non-fat milk for 2 hrs. Subsequently, the membrane was incubated with primary antibody against OPN (1:1000; Chemicon International, Temecula, CA, USA), P2Y1 or P2Y2 (1:300; Abcam, Cambridge, MA, USA), or β-actin (1:1000; Chemicon International). The membranes were then incubated with biotinylated secondary antibody, followed by peroxidase-labeled streptavidin. The signal was captured by chemoluminescence (Pierce Biotechnology). The relative intensity of bands was measured by Scion image analysis software (Scion, Frederick, MD, USA).
Luciferin-Luciferase bioluminescence assay
The extracellular ATP concentration was determined by means of an ENLITEN® ATP Assay System Bioluminescence Detection kit for ATP measurement (Promega, Madison, WI, USA). During analysis, a 100-µL quantity of Enliten Luciferase/Luciferin (L/L) medium (rL/L reagent, reconstitution buffer) was added to 100 µL of sample in the microplate. The resulting light signal was immediately measured by a luminometer (Victor Light Luminescence Counter, PerkinElmer Ltd., Salem, MA, USA). A calibration curve was generated for each luciferase assay by serial dilution of an ATP standard.
Statistical Analysis
All data were analyzed by one-way analysis of variance (ANOVA) with the use of statistical software (SPSS, Chicago, IL, USA). A Scheffé’s test was used for post hoc analysis (p < 0.05).
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RESULTS
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HPDL cells were activated with mechanical stress (1.5 g/cm2) for 24 hrs. The results showed that stress induced the up-regulation of OPN at both mRNA (Fig. 1A ) and protein levels (Fig. 1B). The inductive effect was inhibited by suramin, an antagonist for the P2 receptor family (Fig. 1A), and NF449, an antagonist for P2X1, P2X3, P2Y1, and P2Y2 (Fig. 1B). These results led to the hypothesis that nucleotide receptors, especially P2X1, P2X3, P2Y1, and P2Y2, were involved in the induction of OPN. The band density from Western blot analysis was assessed (Fig. 1).
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Figure 1. Stress-induced OPN expression was inhibited by suramin and NF449. HPDL cells were stimulated with 1.5 g/cm2 of force for 24 hrs. Increased OPN mRNA (a) and protein (b) expression was observed. The stress-induced OPN expression was significantly abolished by both suramin (A) and NF449 (B). The graph represents the band density from Western blot analysis. The results are expressed as mean ± SD from 3 different experiments. *p < 0.05.
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For further analysis of the involvement of nucleotide, the conditioned medium was collected from the culture after application of stress (stress-induced conditioned medium; CM-S) for 2 hrs, transferred to another set of cultures, and culture continued for 24 hrs. The results revealed that the culture incubated with the stress-induced conditioned medium increased OPN expression when compared with that incubated with control conditioned medium (CM-C, Fig. 2A). The results suggested an effect of the secreted molecule(s) from stress-treated cultures in OPN induction.
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Figure 2. The conditioned medium (CM-S) induced the expression of OPN. (A) Cells were incubated for 24 hrs with the medium transferred from the stress-stimulated (CM-S) and non-stimulated (CM-C) cultures. CM-S induced OPN expression when compared with control (CM-C). The inductive effect exerted by CM-S was similar to that resulting from stress (S) and could be inhibited by NF449. (B) The Luciferin-Luciferase bioluminescence assay revealed that the amount of ATP in the medium collected from stress-stimulated cultures (1, 1.5, 2 g/cm2) was increased. (C) Apyrase partially inhibited the CM-S-induced OPN expression. The graph shows the band density from Western blot analysis. The results are expressed as mean ± SD from 3 separate experiments. *p < 0.05; a, mRNA expression; b, protein expression.
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We further examined the amount of ATP in the culture medium and found that stress increased ATP secretion into the medium (Fig. 2B ). The application of apyrase, an ecto-ATPase, could partially inhibit the inductive effect of stress-induced conditioned medium. The data supported the hypothesis that ATP in the stress-induced conditioned medium had a significant effect on OPN induction (Fig. 2C).
Since ATP exerted its effect through the P2 receptor, we investigated the expression of the P2 receptor in HPDL cells. The results indicated that both P2Y1 and P2Y2 receptors were expressed in HPDL cells (Fig. 3A). However, we could not detect the expression of P2X1 and P2X3 receptors (data not shown). To elucidate the kinds of P2Y receptors involved in stress-induced OPN expression, we used MRS2179, a specific P2Y1 antagonist. The results showed that MRS2179 inhibited stress-induced OPN expression in a dose-dependent manner, indicating a role for the P2Y1 receptor (Fig. 3B).
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Figure 3. HPDL cells expressed P2Y1 and P2Y2 ATP receptors. (A) HPDL cells expressed P2Y1 and P2Y2 receptors detectable at both mRNA and protein levels. (B) MRS2179, a specific P2Y1 antagonist, exerted an inhibitory effect on the stress-induced OPN expression in a dose-dependent manner. The graph represents the mean ± SD of the band density from Western blot analysis. The data are from 3 separate experiments. *p < 0.05; a, mRNA expression; b, protein expression.
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To confirm the role of ATP, we applied exogenous ATP (0, 0.1, 1, and 10 µM) to the cultures. We found that ATP induced the expression of OPN in a dose-dependent manner (Fig. 4A). In addition, the inductive effect of ATP was abolished by the Rho kinase inhibitor (Fig. 4B), a result similar to that obtained from mechanical stress application. However, the Rho kinase inhibitor had no effect on the stress-induced release of ATP (Fig. 4C). These results suggested that Rho kinase was involved in the stress/ATP-induced OPN expression, but not in the stress-induced ATP.
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Figure 4. ATP induced the up-regulation of OPN via Rho kinase. (A) HPDL cells were incubated with exogenous ATP (0.1, 1, and 10 µM) for 24 hrs. The results showed that ATP stimulated the expression of OPN in a dose-dependent manner. Cells were incubated with Rho kinase inhibitor (Rhoi) for 30 min before the application of ATP (1 µM) or stress (1.5 g/cm2) for 24 hrs. The results demonstrated that the increase in OPN expression (B), but not that of ATP (C), induced by stress was abolished by Rhoi. The graph in (B) represents the band density from Western blot analysis. The results are expressed as mean ± SD from 3 separate experiments. *p < 0.05; a, mRNA expression; b, protein expression.
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DISCUSSION
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We previously reported that mechanical stress induced OPN expression in HPDL cells (Wongkhantee et al., 2007). In this study, we further demonstrated that the increased expression of OPN was mediated by the release of ATP, which exerted its stimulatory effect through P2Y1 receptors.
An increase in the release of ATP in response to mechanical stress has been shown in several cell types, such as subepithelial fibroblasts (Furuya et al., 2005), chondrocytes (Graff et al., 2000), and osteoblasts (Homolya et al., 2000; Romanello et al., 2001). In the present study, we showed that HPDL cells are able to release ATP in response to mechanical stress. However, the exact mechanism of stress-induced ATP release requires further investigation.
This is the first report showing that ATP induces osteopontin expression. Our previous study showed that mechanical-stress-induced OPN occurred via the Rho kinase pathway. The Rho kinase inhibitor could inhibit the action of ATP-induced OPN, but not the stress-induced ATP release. These findings suggest that stress induces the release of ATP, which in turn mediates Rho kinase activation and results in the up-regulation of OPN. The function of ATP in the conditioned medium was confirmed by apyrase, a potent ecto-ATPase for ATP degradation. However, the incomplete inhibition exerted by apyrase on the conditioned medium-induced OPN expression suggests that there might be other molecule(s) involved in the mechanism, such as other nucleoside triphosphates and diphosphates, which exert their action through the P2Y1 receptor.
We found that HPDL cells expressed both P2Y1 and P2Y2 receptors. Although ATP is able to act through almost all subtypes of P2 receptors (Hoebertz et al., 2003), MRS2179, the specific P2Y1 receptor, could completely inhibit the action of stress-induced OPN. This result indicated that P2Y1 is the main receptor involved in the induction of OPN.
The stimulatory effect of ATP on the expression of OPN could affect the homeostasis of the periodontium. OPN has been shown to facilitate migration and adhesion of osteoclasts (Denhardt and Guo, 1993; Terai et al., 1999). OPN null mice exhibited the lack of bone remodeling (Hoebertz et al., 2000). It is possible that the ATP released from HPDL cells influences the behavior of both osteoblasts and osteoclasts, which express P2 receptors, since ATP can potently enhance the activation and formation of osteoclasts (Morrison et al., 1998) and stimulates cell proliferation in osteoblasts (Orriss et al., 2006). Therefore, the ATP released by HPDL cells may influence the behavior of both PDL and bone cells and subsequently affect the homeostasis of the periodontium.
In conclusion, the present study shows that mechanical stress induces OPN expression via ATP, which mediates the signal through the P2Y1 receptor in HPDL cells. We propose that the increased ATP plays an important role in the mechanism of pressure-induced alveolar bone resorption.
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ACKNOWLEDGMENTS
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This work was supported by the Thailand Research Fund (TRF) and by the Ratchadaphisek Somphot Endowment for Research and the Research Unit, Chulalongkorn University.
Received for publication November 12, 2007.
Revision received February 6, 2008.
Accepted for publication February 12, 2008.
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Journal of Dental Research, Vol. 87, No. 6,
564-568 (2008)
DOI: 10.1177/154405910808700601
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ABSTRACT
INTRODUCTION