© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd
J Periodont Res 2014; 49: 777–784 All rights reserved
JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12162
Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2
J. Manokawinchoke1, A. Pimkhaokhum2, V. Everts3, P. Pavasant1,4 1 Mineralized Tissue Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand, 2Department of Surgery, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand, 3Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute, Amsterdam, The Netherlands and 4 Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Manokawinchoke J, Pimkhaokhum A, Everts V, Pavasant P. Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2. J Periodont Res 2014; 49: 777–784. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Prostaglandin E2 (PGE2) has been shown to be able to influence both bone formation and resorption. The purpose of this study was to investigate the effect of PGE2 on the osteogenic differentiation of human periodontal ligament (HPDL) cells. Material and Methods: HPDL cells were cultured with 0.001–1 lM PGE2 in osteogenic medium. In-vitro mineral deposition was determined by Alizarin Red S staining, and gene expression was determined by real-time PCR. Results: PGE2 inhibited in-vitro mineral deposition by HPDL cells in a dosedependent manner. PCR analyses showed that PGE2 upregulated the expression of Runt-related transcription factor 2 (RUNX2), but had no effect on osteocalcin expression. Upregulation of TWIST-related protein1 (TWIST1), a functional antagonist of RUNX2, was also observed. In addition, increased levels of RUNX2 and TWIST1 proteins, induced by PGE2, were detected by western blot analysis. Using a chemical activator of E prostanoid (EP) receptors as well as small interfering RNA against an EP receptor, it was shown that PGE2 regulated RUNX2 and TWIST1 via the EP2 receptor. The role of protein kinase A in the inductive effect of PGE2 was also demonstrated. Conclusion: The results of this study revealed that PGE2 modulates the osteogenic differentiation of HPDL cells via regulating the expression of RUNX2 and TWIST1. The results suggest a possible role for PGE2 in regulating the homeostasis of periodontal ligament tissue.
Prostaglandin E2 (PGE2) is a lipid mediator that plays a role in regulating the inflammatory process and is
also involved in a wide range of physiological activities in our body, such as in the cardiovascular, endocrine,
Prasit Pavasant, DDS, PhD, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Rd. Pathumwan, Bangkok 10330, Thailand Tel: +66 2 218 8872 Fax: +66 2 218 8870 e-mail: [email protected] Key words: human periodontal ligament cells;
in-vitro mineral deposition; PGE2; RUNX2; TWIST1 Accepted for publication November 30, 2013
gastrointestinal, neural and reproductive systems (1,2). PGE2 has been demonstrated to play a role in cell
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proliferation and cell differentiation, and in epithelial, endothelial, mesenchymal and immune cell functions (3–6). In addition, this mediator participates in hard-tissue homeostasis. The effect of PGE2 on bone resorption occurs through the upregulation of RANKL, a key molecule that regulates osteoclast differentiation, activation and survival (7). Besides resorption, evidence also suggests that PGE2 functions in bone formation and osteogenic differentiation by mesenchymal stem cells (8–10). The actions of PGE2 are mediated by activating cell-surface receptors. There are at least four subtypes of cell-surface receptors for prostaglandin E, namely E prostanoid (EP) 1–4, which are expressed in a tissue- and cell-specific manner (11). The EP2 and EP4 subtypes have been shown to play roles in osteoblast function and differentiation. In addition, both EP2 and EP4 are involved in the regulation of the cAMP level (10,12,13). PGE2 can also increase the proliferation, and inhibit apoptosis, of bone marrow stromal cells (14). The addition of PGE2 or an EP2/EP4 agonist was reported to stimulate osteoblast differentiation in vitro (14,15). Furthermore, exogenous PGE2 induced osteogenic differentiation by human tendon stem cells through the induction of bone morphogenetic protein 2 (16). The role of PGE2 in bone formation has also been shown in animal models (17). Furthermore, both local and systemic applications of EP2 or EP4 agonists showed an anabolic effect on bone formation (18–21). However, the underlying mechanism of how PGE2 promotes osteogenic differentiation is still largely unclear. Runt-related transcription factor 2 (RUNX2), formerly known as Corebinding factor alpha1 (CBFA1), is a key transcription factor associated with osteoblast differentiation. This protein is a member of the RUNX family of transcription factors that possess a Runt DNA-binding domain. RUNX2 is essential for osteoblastic differentiation, skeletal morphogenesis and the expression of regulatory factors involved in skeletal gene expression
(22,23). Mineralized bone does not develop in RUNX2 null mice, indicating the importance of this protein in bone formation (22,23). The function of RUNX2 is counteracted by TWIST proteins (24,25). TWIST-related proteins are basic helix–loop–helix transcription factors that consist of TWIST-related protein1 (TWIST1) and TWIST-related protein2 (TWIST2) or Dermo1. TWIST1 and TWIST2 inhibit osteoblast maturation and maintain cells in a preosteoblastic phenotype (24). TWIST has been proposed to function by directly interacting with RUNX2 and by inhibiting osteogenic induction by RUNX2 (24,25). Therefore, TWIST is considered to be an inhibitor of osteogenic differentiation. Genetic mutations in the human TWIST gene that cause TWIST haploinsufficiency and premature cranial suture fusion (26,27) as a result of increased bone formation have been identified in Saethre–Chotzen syndrome (28). In-vitro molecular silencing of TWIST1 in murine mesenchymal stem cells promoted osteoblast gene expression and matrix mineralization (29). Consistent with these findings, reduction of TWIST1 by antisense oligonucleotides increased bone cell maturation (30), whereas over-expression of TWIST1 in osteoblasts attenuated osteogenic differentiation (31). Our previous results showed that HPDL cells, upon activation by mechanical stress or ATP, increased the expression of RANKL, a key modulator of osteoclastogenesis, through a PGE2-dependent pathway (32,33). PGE2-induced expression of RANKL has been proposed to participate in periodontal remodeling and inflammation by regulating the release of inflammatory cytokines that play a role in osteoclastogenesis (32,34). Although the role of PGE2 has been demonstrated in inflammation and the regulation of osteoclast formation, the involvement of PGE2 in the osteogenic differentiation of human periodontal ligament (HPDL) cells is still unknown. In the present study, the influence of PGE2 on the osteogenic differentiation of HPDL cells was investigated
and we also examined the underlying mechanism. The results may help to clarify the role of PGE2 in the homeostasis of the periodontium.
Material and methods Cell culture
HPDL cells were obtained from healthy periodontal ligament tissue of noncarious, freshly extracted third molars removed for orthodontic reasons. The protocol was approved by the Ethical Committee, Faculty of Dentistry, Chulalongkorn University. Informed consent was obtained from each patient. Briefly, teeth were rinsed with sterile phosphate-buffered saline. The periodontal ligament tissue was removed from the middle third of the root, harvested on 60-mm culture dishes and cultured in growth medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ mL of penicillin, 100 lg/mL of streptomycin and 5 lg/mL of amphotericin B) in a humidified atmosphere of 95% air, 5% CO2 at 37°C. Cells from passage 3 from three different donors were used in each experiment, which were performed in triplicate. Media and supplements were obtained from Gibco BRL (Carlsbad, CA, USA). PGE2 incubation
HPDL cells were seeded in 12-well plates at a density of 37,500 cells/cm2 and incubated for 24 h. Cells were starved in serum-free medium for 8 h and then the medium was replaced with either growth or osteogenic medium. The latter medium consisted of growth medium supplemented with 50 lg/mL of ascorbic acid, 250 nM dexamethasone and 5 mM beta-glycerophosphate (Sigma-Aldrich Chemical, St Louis, MO, USA). Cells were cultured with 0.001–1 lM prostaglandin E2 (Cayman Chemical, Ann Arbor, MI, USA), 0.001–1 lM butaprost, an EP2 agonist (Cayman Chemical), 0.6–42 nM sulprostone, an EP1 and EP3 agonist (Tocris Bioscience, Ellisville, MO, USA) or 1.25–2.5 nM TCS2510, an EP4 agonist (Tocris Bioscience).
PGE2 suppresses in-vitro calcification in HPDL cells For the protein kinase A (PKA) signaling pathway experiments, cells were cultured with 20 lM of a PKA inhibitor (adenosine 3′,5′-cyclic monophosphorothioate, Rp-Isomer, triethylammonium salt; Calbiochem, EMD Biosciences, Inc., San Diego, CA, USA) for 30 min before stimulation with PGE2. After culture for 24 h, total RNA was extracted and subjected to quantitative real-time PCR (qPCR; see below). Cellular protein levels were determined by western blot analysis. The most effective dose of PGE2 was selected and used for the rest of the experiments. Transfection of small interfering RNA
HPDL cells were grown in six-well plates and cultured in medium, without antibiotics, to 70–80% confluence. Cells were then treated, according to the manufacturer’s instructions, with a solution of small interfering (si) RNA oligonucleotides specific to EP2 (Santa Cruz Biotechnology, Dallas, TX, USA). Control siRNA (Santa Cruz Biotechnology) was used as a control treatment. Transfection with the siRNA was performed 24 h before exposure to PGE2. After 24 h of treatment with PGE2, mRNA was isolated for further analysis. Osteogenic differentiation
Cells were seeded at the same density, as described above, in a 24-well plate and were maintained in osteogenic medium. The medium was changed
every 48 h. Osteoblast marker gene expression and mineral deposition was investigated using the methods described below. Quantitative real-time PCR analysis
Total RNA was extracted using IsolRNA Lysis Reagent (5Prime, Gaitherburg, MD, USA) according to the manufacturer’s instructions. One microgram of each RNA sample was converted to cDNA by avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) for 1.5 h at 42°C. Subsequently, qPCR was performed in a Lightcycler Nano real-time PCR system (Roche Applied Science, Indianapolis, IN, USA) using the SYBR Green system (Roche Applied Science). Amplification was performed as follows: 40 cycles of denaturation at 94°C for 20 s, annealing at 60°C for 20 s and extension at 72°C for 20 s. The reaction product was quantified with glyceraldehyde3-phosphate dehydrogenase (GAPDH) as the reference gene. The primers were designed based on the sequences reported in GenBank and are shown in Table 1. Mineralization assay
The cells were fixed with cold methanol for 10 min, washed with deionized water and stained with 1% Alizarin Red S solution for 3 min at room temperature. To quantify the amount of calcium deposited, the stain was eluted with 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate at room temperature for
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15 min and the absorbance was read at 570 nm. Protein extraction and western blot analysis
Protein was extracted with radioimmunoprecipitation buffer (50 mM Tris/ HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% Na-deoxycholate) containing a cocktail of protease inhibitors and phosphatase inhibitors (1 mM sodium vanadate, 50 mM NaF; SigmaAldrich Chemical). Protein concentrations were measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA), and equal amounts of protein samples were electrophoresed through a 12% sodium dodecyl sulfate–polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane. The membrane was incubated with primary antibody against TWIST1 (dilution 1 : 250; Abcam, Cambridge, UK), RUNX2 (dilution 1 : 1000; R & D Systems, Minneapolis, MN, USA) or GAPDH (dilution 1 : 2000; Chemicon International, Temecula, CA, USA) at 4°C. The membranes were then incubated with biotinylated secondary antibody, followed by peroxidase-labeled streptavidin. The signal was detected by chemiluminescence (Pierce Biotechnology) and quantified using an image analyzer (Vilber Lourmat, Marne-la-Vallee, France). Statistical analysis
All data were analyzed by one-way ANOVA using statistical software (SPSS, Chicago, IL, USA). Scheffe’s
Table 1. Primer sequences used for quantitative real-time PCR analysis
Gene symbol
Forward (5′–3′)
Reverse (5′–3′)
Accession number
RUNX2 (OSF2/ CBFA1a or RUNX2 type II) OCN TWIST1 TWIST2 GAPDH
ATG ATG ACA CTG CCA CCT CTG A
GGC TGG ATA GTG CAT TCG TG
NM_057179.2
CTT TGT GTC CAA GCA GGA GG TCT TAC GAG GAG CTG CAG ACG CA GCT GCG CAA GAT CAT CCC TCA TGG GTG TGA ACC ATG AGA A
CTG AAA GCC GAT GTG GTC AG ATC TTG GAG TCC AGC TCG TCG CT GTA GCT GCA GCT GGT CAT C GGC ATG GAC TGT GGT CAT GAG
NM_199173.4 NM_000474.3 NM_033012.2 NM_002046.3
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OCN, osteocalcin; RUNX2, Runt-related transcription factor 2; TWIST1, TWISTrelated protein1; TWIST2, TWIST-related protein2.
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test was used for post-hoc analysis ( p < 0.05).
Results In the first experiment, HPDL cells were cultured in osteogenic medium with or without PGE2 ranging in concentration from 0.001 to 1 lM. None of the concentrations of PGE2 used were toxic to the cells after 3 d in culture, as indicated by the MTT assay (data not shown). Osteogenic differentiation was determined based on in-vitro mineral deposition using Alizarin Red S staining. The results in Fig. 1 indicate that the addition of exogenous PGE2 to the cultures suppressed the in-vitro mineral deposition by HPDL cells in a dose-dependent manner. The amount of calcium deposited was quantified by eluting the stain with 10% cetylpyridinium chloride monohydrate and assessing the absorbance at 570 nm (Fig. 1). The quantitative results supported the decrease in calcium staining and a
significant inhibitory effect was found at concentrations of 0.01 lM PGE2 and higher. Our preliminary results indicated that the peak expression of mRNA for osteogenic markers in HPDL cells cultured in osteogenic medium occurred at day 10 (data not shown). Therefore, this time point was chosen to examine further the influence of PGE2 on osteogenesis in HPDL cells (Fig. 2). Interestingly, a dose-dependent upregulation of RUNX2 by PGE2 was observed, despite the inhibitory effect of PGE2 on mineral deposition. The expression of osteocalcin, a RUNX2 target gene and a marker of osteogenic differentiation, did not significantly change when exposed to PGE2. In addition, the results revealed increased expression of TWIST1, an inhibitor of RUNX2, but no effect on the expression of TWIST2, demonstrating that PGE2 induced TWIST1 expression. The inductive effect of PGE2 on TWIST1 expression was apparent at 0.01 lM PGE2 or higher.
Fig. 1. Prostaglandin E2 (PGE2) inhibits the osteogenic differentiation of human periodontal ligament (HPDL) cells in vitro. HPDL cells were cultured in osteogenic medium in the presence of 0.001–1 lM PGE2 for 18 d (D18). In-vitro mineral deposition was determined by Alizarin Red S staining. The bar chart shows the staining quantified by eluting the stain with 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate at room temperature for 15 min followed by reading the absorbance at 570 nm. The staining at day 0 (D0) was used as background. The results represent the average + SD from three experiments. Asterisks indicate statistical significance at p < 0.05. The experiments were performed in triplicate using HPDL cell lines from three donors.
Next, we tested the short-term influence of PGE2 on HPDL cells to determine the direct influence of PGE2 on osteogenic-related gene expression. The results shown in Fig. 3 indicate that PGE2 increased the expression of RUNX2 and TWIST1 mRNAs within the first 24 h. In addition, analysis of the levels of RUNX2 and TWIST1 proteins was performed at 1, 2, 3 and 4 d (data not shown). However, an obvious increase was noted only at day 4 of treatment with PGE2 (Fig. 3). HPDL cells expressed all known EP receptors (Figure S1). To investigate the possible role of the EP receptors, cells were treated with EP agonists and the expression of RUNX2 and TWIST1 was examined by qPCR (Fig. 4A). Among the EP agonists used, only the EP2 agonist significantly increased the expression of RUNX2 and TWIST1. Furthermore, the increased expression of RUNX2 and TWIST1 induced by the EP2 agonist was found to be dosedependent, as shown in Fig. 4B. Gene silencing using siRNA was performed to analyze the possible role of the EP2 receptor. Transfection with siEP2 inhibited the expression of EP2 by more than 70% (Figure S2). The results from Fig. 4C reveal that the inductive effect of PGE2 on RUNX2 and TWIST1 expression was attenuated in HPDL cells transfected with siEP2. Because the EP receptor is associated with the cAMP–PKA signaling pathway, the involvement of PKA in the inductive effect of PGE2 was investigated. Cells were cultured with PGE2 in the presence or absence of a PKA inhibitor. The results indicated that the PKA inhibitor attenuates the inductive effect of PGE2 on TWIST1 expression (Fig. 5A). In addition, the application of forskolin, a PKA activator, up-regulated TWIST1 in a dose-dependent manner (Fig. 5B), in contrast to the effect of the PKA inhibitor.
Discussion The present study shows, for the first time, that PGE2 can modulate the
PGE2 suppresses in-vitro calcification in HPDL cells
Fig. 2. Prostaglandin E2 (PGE2) induces Runt-related transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1) expression by human periodontal ligament (HPDL) cells. Cells were treated with 0.001–1 lM PGE2 for 10 d. The expression of mRNAs for RUNX2, osteocalcin (OCN) and TWIST1 and TWIST-related protein2 (TWIST2) was analyzed by real-time PCR and the results are shown as relative expression compared with the control culture without PGE2. The level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The results represent the average + SD from three experiments. Asterisks indicate statistical significance at p < 0.05. The experiments were performed in triplicate using HPDL cell lines from three donors.
A
B
C
Fig. 3. Prostaglandin E2 (PGE2) increases both protein and mRNA expression of Runtrelated transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1). The cells were treated with 0.001–1 lM PGE2 for 1 d. The expression of mRNA for RUNX2 (A) and for TWIST1 and TWIST-related protein2 (TWIST2) (B) was detected by real-time PCR and the results are shown as relative expression compared with the control culture without PGE2, with the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the internal control. The results represent the average + SD from three experiments. Asterisks indicate statistical significance at p < 0.05. Western blot analysis was performed to confirm the result from PCR analysis (C). The experiments were performed in triplicate using human periodontal ligament (HPDL) cell lines from three donors.
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expression of both RUNX2 and TWIST1, two key genes involved in the regulation of osteogenic differentiation, in HPDL cells. Despite inducing an increase in the expression of RUNX2 mRNA and RUNX2 protein, PGE2 inhibited in-vitro mineral deposition by HPDL cells cultured in osteogenic medium. This inhibitory effect might be caused by the upregulation of TWIST1, an inhibitor of RUNX2. The inductive effect of PGE2 on both genes was found to depend on the EP2–PKA signaling pathway. Periodontal ligament cells reside within the periodontal ligament that functions as a cushion to absorb the masticatory forces exerted upon the tooth. These cells possess several osteoblastic characteristics, such as the expression of alkaline phosphatase and the ability to generate in-vitro mineral deposition. We previously reported that periodontal ligament cells responded to applied pressure by releasing PGE2 (32,33). The results of the present study suggest a role for PGE2 in the homeostasis of the periodontal ligament. PGE2 might help to maintain the fibroblastic phenotype and prevent or inhibit the osteogenic differentiation of HPDL cells. PGE2 has been shown to exert both anabolic and catabolic effects on bone formation. The inductive effect of PGE2 on osteoclast formation has been reported (34). PGE2 is a potent inducer of RANKL, which plays an essential role in osteoclastogenesis via its interaction with RANK on the osteoclast precursor surface. Moreover, it was reported that PGE2 may play a role in bone formation (17). PGE2 has been shown to promote the proliferation and differentiation of marrow stromal cells in vitro (14) and induced cancellous bone formation in rats (17). However, in contrast to HPDL cells, PGE2 induced RUNX2 expression and in-vitro mineral deposition by rat marrow stromal cells (21). The difference in the results is probably caused by the different cell types and species used in these studies. Our previous results also indicated different responses between HPDL cells and mouse marrow stromal cells (35). In that study, capsaicin induced osteoprotegerin expression
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A
B
C
Fig. 4. E prostanoid receptor 2 (EP2) involvement in prostaglandin E2 (PGE2)-induced Runt-related transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1) expression. Cells were treated with EP1–4 agonists and the expression of RUNX2 and TWIST1 were determined by real-time PCR (A). Various doses of EP2 agonist were used to further confirm the effect on expression of RUNX2 and TWIST1 mRNAs (B). EP2 silencing using small interfering RNA (siRNA) abolished the inductive effect of PGE2 on RUNX2 and TWIST1 expression (C). The results are shown as relative expression from three experiments using human periodontal ligament (HPDL) cells from three donors.
in HPDL cells, whereas it induced RANKL expression in mouse marrow stromal cells. The exact mechanisms underlying these differences are still unclear. PGE2 exerts its effect via EP receptors. Among the four subtypes of EP receptor, EP2 and EP4 are the main receptors that participate in mineralized tissue homeostasis. The application of EP2 and EP4 agonists has been demonstrated to either induce or inhibit bone formation (21,36–39). Therefore, the exact role of each of these two receptors remains unclear. It has been reported that PGE2 induces RUNX2 expression and mineralization via EP2 and EP4 in rat and mouse bone marrow cultures (21,39). In addition, EP2 and EP4 agonists increased bone formation in an
animal model (21,36). EP2 and EP4 have also been shown to participate in PGE2-induced bone resorption (37,38,40). The mechanisms underlying the anabolic or catabolic effects of EP2 and EP4 in bone homeostasis are as yet unknown. It is possible that EP receptors have functional redundancy and that the chemical agonist used in those experiments might activate more than one EP receptor. In the present study, the EP4 agonist did not have any effect on the expression of RUNX2 or TWIST1. The application of the EP2 agonist, however, showed an effect similar to that seen with PGE2. The role of EP2 was confirmed by the use of a silencing approach, indicating the importance of EP2 in HPDL cells in their response to PGE2.
It has been demonstrated that the EP2 receptor activated by PGE2 resulted in an increased level of cytosolic cAMP (41,42). Subsequent to its formation, cAMP can either activate adenylate kinase or translocate into the nucleus. In the present study, PKA inhibition reduced the expression of RUNX2 and TWIST1, suggesting involvement of the EP2–PKA pathway. The role of EP2–PKA was reported for different biological processes, such as mast cell degranulation or interleukin-8 expression by colonic epithelial cells (41,42). Whether this pathway is involved in RUNX2 and TWIST1 expression in other cell types needs further investigation. RUNX2 (or CBFA1) is a key regulator of osteogenic differentiation (22,23). In RUNX2 knockout mice, mineralized bone is absent because of a defect in osteoblast differentiation (22). Our finding, that PGE2 upregulates RUNX2, suggests that the anabolic effect of PGE2 might occur via this induction. Interestingly, we found that, despite increased expression of RUNX2, in-vitro mineral deposition was inhibited. It is possible that PGE2 exerted this inhibitory effect via the upregulation of TWIST1 because TWIST1 has been demonstrated to be able to bind to RUNX2 and inhibit its function. Although TWIST inhibited the function of RUNX2, it had no effect on RUNX2 expression (24). Moreover, in TWIST1+/ mice, premature osteoblast differentiation and premature trabecular bone formation was detected in embryonic day 16 embryos (24). The inhibitory role of TWIST1 coincides with the observation that the expression of osteocalcin was not increased. As osteocalcin is one of the target molecules of RUNX2, the explanation could be that TWIST1 binds to RUNX2 and thereby prevents the function of this transcription factor. In conclusion, PGE2 was shown to regulate the expression of RUNX2 and TWIST1 mRNAs and RUNX2 and TWIST1 proteins. Our data strongly suggest the involvement of the EP2–PKA signaling pathway in this process. The balance between RUNX2 and TWIST1 may regulate
PGE2 suppresses in-vitro calcification in HPDL cells A
B
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Fig. 5. The inductive effect of prostaglandin E2 (PGE2) occurs via the protein kinase A (PKA) pathway. Cells were treated with 1 lM PGE2, and the expression of Runt-related transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1) was determined by real-time PCR. Application of a PKA inhibitor (PKAi) inhibited the inductive effect of PGE2. Moreover, when cells were treated with forskolin, a PKA activator, upregulation of RUNX2 and TWIST1 was observed.
the differentiation state of PDL cells. The level and duration of exposure to PGE2 may help to regulate the behavior of PDL cells and help to modulate the homeostasis of periodontal tissue.
Acknowledgements This work was supported by “Integrated Innovation Academic Center: IIAC” Chulalongkorn University Centenary Academic Development Project (CU05_55_32) and the Research Chair Grant of the National Science and Technology Development Agency.
Source of funding Integrated Innovation Academic Center (IIAC), Chulalongkorn University Centenary Academic Development Project; The National Science and Technology Development Agency (NSTDA) Research Chair Grant.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Human PDL cells express EP receptors.
Figure S2 siRNA reduced EP2 expression.
application
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References 1. Akaogi J, Nozaki T, Satoh M, Yamada H. Role of PGE2 and EP receptors in the pathogenesis of rheumatoid arthritis and as a novel therapeutic strategy. Endocr Metab Immune Disord Drug Targets 2006;6:383–394. 2. Kalinski P. Regulation of immune responses by prostaglandin E2. J Immunol 2012;188:21–28. 3. Aronoff DM, Canetti C, Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP. J Immunol 2004;173:559–565. 4. Huang S, Wettlaufer SH, Hogaboam C, Aronoff DM, Peters-Golden M. Prostaglandin E(2) inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling. Am J Physiol Lung Cell Mol Physiol 2007;292:L405–L413. 5. Kolodsick JE, Peters-Golden M, Larios J, Toews GB, Thannickal VJ, Moore BB. Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation. Am J Respir Cell Mol Biol 2003;29:537–544. 6. White ES, Atrasz RG, Dickie EG et al. Prostaglandin E(2) inhibits fibroblast
13.
14.
15.
16.
17.
18.
783
migration by E-prostanoid 2 receptormediated increase in PTEN activity. Am J Respir Cell Mol Biol 2005;32:135–141. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–342. Coetzee M, Haag M, Kruger MC. Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3-E1 osteoblastlike cells. Cell Biochem Funct 2009;27: 3–11. Hagino H, Kuraoka M, Kameyama Y, Okano T, Teshima R. Effect of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the cortical bone response to mechanical loading. Bone 2005;36:444–453. Minamizaki T, Yoshiko Y, Kozai K, Aubin JE, Maeda N. EP2 and EP4 receptors differentially mediate MAPK pathways underlying anabolic actions of prostaglandin E2 on bone formation in rat calvaria cell cultures. Bone 2009;44: 1177–1185. Breyer RM, Bagdassarian CK, Myers SA, Breyer MD. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 2001;41:661–690. Alander CB, Raisz LG. Effects of selective prostaglandins E2 receptor agonists on cultured calvarial murine osteoblastic cells. Prostaglandins Other Lipid Mediat 2006;81:178–183. Paralkar VM, Borovecki F, Ke HZ et al. An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci U S A 2003;100: 6736–6740. Weinreb M, Shamir D, Machwate M, Rodan GA, Harada S, Keila S. Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity. Prostaglandins Leukot Essent Fatty Acids 2006;75:81–90. Raisz LG, Woodiel FN. Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures. Prostaglandins Other Lipid Mediat 2003;71: 287–292. Zhang J, Wang JH. BMP-2 mediates PGE(2) -induced reduction of proliferation and osteogenic differentiation of human tendon stem cells. J Orthop Res 2012;30:47–52. Keila S, Kelner A, Weinreb M. Systemic prostaglandin E2 increases cancellous bone formation and mass in aging rats and stimulates their bone marrow osteogenic capacity in vivo and in vitro. J Endocrinol 2001;168:131–139. Ke HZ, Crawford DT, Qi H et al. A nonprostanoid EP4 receptor selective
784
19.
20.
21.
22.
23.
24.
25.
26.
27.
Manokawinchoke et al.
prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats. J Bone Miner Res 2006;21:565–575. Li M, Thompson DD, Paralkar VM. Prostaglandin E(2) receptors in bone formation. Int Orthop 2007;31:767–772. Tanaka M, Sakai A, Uchida S et al. Prostaglandin E2 receptor (EP4) selective agonist (ONO-4819.CD) accelerates bone repair of femoral cortex after drill-hole injury associated with local upregulation of bone turnover in mature rats. Bone 2004;34:940–948. Yoshida K, Oida H, Kobayashi T et al. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc Natl Acad Sci U S A 2002;99:4580–4585. Komori T, Yagi H, Nomura S et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755–764. Otto F, Thornell AP, Crompton T et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765–771. Bialek P, Kern B, Yang X et al. A twist code determines the onset of osteoblast differentiation. Dev Cell 2004;6:423–435. Yousfi M, Lasmoles F, Marie PJ. TWIST inactivation reduces CBFA1/ RUNX2 expression and DNA binding to the osteocalcin promoter in osteoblasts. Biochem Biophys Res Commun 2002;297:641–644. el Ghouzzi V, Le Merrer M, PerrinSchmitt F et al. Mutations of the TWIST gene in the Saethre-Chotzen syndrome. Nat Genet 1997;15:42–46. Howard TD, Paznekas WA, Green ED et al. Mutations in TWIST, a basic helixloop-helix transcription factor, in
28.
29.
30.
31.
32.
33.
34.
Saethre-Chotzen syndrome. Nat Genet 1997;15:36–41. Yousfi M, Lasmoles F, Lomri A, Delannoy P, Marie PJ. Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome. J Clin Investig 2001;107:1153–1161. Miraoui H, Severe N, Vaudin P, Pages JC, Marie PJ. Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells: implication of FGFR2 signaling. J Cell Biochem 2010;110:1147–1154. Lee MS, Lowe GN, Strong DD, Wergedal JE, Glackin CA. TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage. J Cell Biochem 1999;75:566–577. Danciu TE, Li Y, Koh A, Xiao G, McCauley LK, Franceschi RT. The basic helix loop helix transcription factor Twist1 is a novel regulator of ATF4 in osteoblasts. J Cell Biochem 2012;113: 70–79. Luckprom P, Wongkhantee S, Yongchaitrakul T, Pavasant P. Adenosine triphosphate stimulates RANKL expression through P2Y1 receptor-cyclo-oxygenasedependent pathway in human periodontal ligament cells. J Periodontal Res 2010;45:404–411. Wongkhantee S, Yongchaitrakul T, Pavasant P. Mechanical stress induces osteopontin expression in human periodontal ligament cells through rho kinase. J Periodontol 2007;78:1113–1119. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res 2002;17:210–220.
35. Sooampon S, Manokawinchoke J, Pavasant P. Transient receptor potential vanilloid-1 regulates osteoprotegerin/ RANKL homeostasis in human periodontal ligament cells. J Periodontal Res 2013;48:22–29. 36. Kambe T, Maruyama T, Nakai Y et al. Discovery of novel prostaglandin analogs as potent and selective EP2/EP4 dual agonists. Bioorg Med Chem 2012;20: 2235–2251. 37. Sakuma Y, Tanaka K, Suda M et al. Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide. J Bone Miner Res 2000;15:218–227. 38. Suzawa T, Miyaura C, Inada M et al. The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs. Endocrinology 2000;141:1554–1559. 39. Weinreb M, Grosskopf A, Shir N. The anabolic effect of PGE2 in rat bone marrow cultures is mediated via the EP4 receptor subtype. Am J Physiol 1999;276: E376–E383. 40. Li X, Okada Y, Pilbeam CC et al. Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro. Endocrinology 2000;141:2054–2061. 41. Serra-Pages M, Olivera A, Torres R, Picado C, de Mora F, Rivera J. E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling. J Leukoc Biol 2012;92:1155–1165. 42. Srivastava V, Dey I, Leung P, Chadee K. Prostaglandin E2 modulates IL-8 expression through formation of a multiprotein enhanceosome in human colonic epithelial cells. Eur J Immunol 2012;42:912–923.
J Periodont Res 2014; 49: 777–784 All rights reserved
JOURNAL OF PERIODONTAL RESEARCH doi:10.1111/jre.12162
Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2
J. Manokawinchoke1, A. Pimkhaokhum2, V. Everts3, P. Pavasant1,4 1 Mineralized Tissue Research Unit, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand, 2Department of Surgery, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand, 3Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute, Amsterdam, The Netherlands and 4 Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand
Manokawinchoke J, Pimkhaokhum A, Everts V, Pavasant P. Prostaglandin E2 inhibits in-vitro mineral deposition by human periodontal ligament cells via modulating the expression of TWIST1 and RUNX2. J Periodont Res 2014; 49: 777–784. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Background and Objective: Prostaglandin E2 (PGE2) has been shown to be able to influence both bone formation and resorption. The purpose of this study was to investigate the effect of PGE2 on the osteogenic differentiation of human periodontal ligament (HPDL) cells. Material and Methods: HPDL cells were cultured with 0.001–1 lM PGE2 in osteogenic medium. In-vitro mineral deposition was determined by Alizarin Red S staining, and gene expression was determined by real-time PCR. Results: PGE2 inhibited in-vitro mineral deposition by HPDL cells in a dosedependent manner. PCR analyses showed that PGE2 upregulated the expression of Runt-related transcription factor 2 (RUNX2), but had no effect on osteocalcin expression. Upregulation of TWIST-related protein1 (TWIST1), a functional antagonist of RUNX2, was also observed. In addition, increased levels of RUNX2 and TWIST1 proteins, induced by PGE2, were detected by western blot analysis. Using a chemical activator of E prostanoid (EP) receptors as well as small interfering RNA against an EP receptor, it was shown that PGE2 regulated RUNX2 and TWIST1 via the EP2 receptor. The role of protein kinase A in the inductive effect of PGE2 was also demonstrated. Conclusion: The results of this study revealed that PGE2 modulates the osteogenic differentiation of HPDL cells via regulating the expression of RUNX2 and TWIST1. The results suggest a possible role for PGE2 in regulating the homeostasis of periodontal ligament tissue.
Prostaglandin E2 (PGE2) is a lipid mediator that plays a role in regulating the inflammatory process and is
also involved in a wide range of physiological activities in our body, such as in the cardiovascular, endocrine,
Prasit Pavasant, DDS, PhD, Department of Anatomy, Faculty of Dentistry, Chulalongkorn University, Henri-Dunant Rd. Pathumwan, Bangkok 10330, Thailand Tel: +66 2 218 8872 Fax: +66 2 218 8870 e-mail: [email protected] Key words: human periodontal ligament cells;
in-vitro mineral deposition; PGE2; RUNX2; TWIST1 Accepted for publication November 30, 2013
gastrointestinal, neural and reproductive systems (1,2). PGE2 has been demonstrated to play a role in cell
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proliferation and cell differentiation, and in epithelial, endothelial, mesenchymal and immune cell functions (3–6). In addition, this mediator participates in hard-tissue homeostasis. The effect of PGE2 on bone resorption occurs through the upregulation of RANKL, a key molecule that regulates osteoclast differentiation, activation and survival (7). Besides resorption, evidence also suggests that PGE2 functions in bone formation and osteogenic differentiation by mesenchymal stem cells (8–10). The actions of PGE2 are mediated by activating cell-surface receptors. There are at least four subtypes of cell-surface receptors for prostaglandin E, namely E prostanoid (EP) 1–4, which are expressed in a tissue- and cell-specific manner (11). The EP2 and EP4 subtypes have been shown to play roles in osteoblast function and differentiation. In addition, both EP2 and EP4 are involved in the regulation of the cAMP level (10,12,13). PGE2 can also increase the proliferation, and inhibit apoptosis, of bone marrow stromal cells (14). The addition of PGE2 or an EP2/EP4 agonist was reported to stimulate osteoblast differentiation in vitro (14,15). Furthermore, exogenous PGE2 induced osteogenic differentiation by human tendon stem cells through the induction of bone morphogenetic protein 2 (16). The role of PGE2 in bone formation has also been shown in animal models (17). Furthermore, both local and systemic applications of EP2 or EP4 agonists showed an anabolic effect on bone formation (18–21). However, the underlying mechanism of how PGE2 promotes osteogenic differentiation is still largely unclear. Runt-related transcription factor 2 (RUNX2), formerly known as Corebinding factor alpha1 (CBFA1), is a key transcription factor associated with osteoblast differentiation. This protein is a member of the RUNX family of transcription factors that possess a Runt DNA-binding domain. RUNX2 is essential for osteoblastic differentiation, skeletal morphogenesis and the expression of regulatory factors involved in skeletal gene expression
(22,23). Mineralized bone does not develop in RUNX2 null mice, indicating the importance of this protein in bone formation (22,23). The function of RUNX2 is counteracted by TWIST proteins (24,25). TWIST-related proteins are basic helix–loop–helix transcription factors that consist of TWIST-related protein1 (TWIST1) and TWIST-related protein2 (TWIST2) or Dermo1. TWIST1 and TWIST2 inhibit osteoblast maturation and maintain cells in a preosteoblastic phenotype (24). TWIST has been proposed to function by directly interacting with RUNX2 and by inhibiting osteogenic induction by RUNX2 (24,25). Therefore, TWIST is considered to be an inhibitor of osteogenic differentiation. Genetic mutations in the human TWIST gene that cause TWIST haploinsufficiency and premature cranial suture fusion (26,27) as a result of increased bone formation have been identified in Saethre–Chotzen syndrome (28). In-vitro molecular silencing of TWIST1 in murine mesenchymal stem cells promoted osteoblast gene expression and matrix mineralization (29). Consistent with these findings, reduction of TWIST1 by antisense oligonucleotides increased bone cell maturation (30), whereas over-expression of TWIST1 in osteoblasts attenuated osteogenic differentiation (31). Our previous results showed that HPDL cells, upon activation by mechanical stress or ATP, increased the expression of RANKL, a key modulator of osteoclastogenesis, through a PGE2-dependent pathway (32,33). PGE2-induced expression of RANKL has been proposed to participate in periodontal remodeling and inflammation by regulating the release of inflammatory cytokines that play a role in osteoclastogenesis (32,34). Although the role of PGE2 has been demonstrated in inflammation and the regulation of osteoclast formation, the involvement of PGE2 in the osteogenic differentiation of human periodontal ligament (HPDL) cells is still unknown. In the present study, the influence of PGE2 on the osteogenic differentiation of HPDL cells was investigated
and we also examined the underlying mechanism. The results may help to clarify the role of PGE2 in the homeostasis of the periodontium.
Material and methods Cell culture
HPDL cells were obtained from healthy periodontal ligament tissue of noncarious, freshly extracted third molars removed for orthodontic reasons. The protocol was approved by the Ethical Committee, Faculty of Dentistry, Chulalongkorn University. Informed consent was obtained from each patient. Briefly, teeth were rinsed with sterile phosphate-buffered saline. The periodontal ligament tissue was removed from the middle third of the root, harvested on 60-mm culture dishes and cultured in growth medium (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ mL of penicillin, 100 lg/mL of streptomycin and 5 lg/mL of amphotericin B) in a humidified atmosphere of 95% air, 5% CO2 at 37°C. Cells from passage 3 from three different donors were used in each experiment, which were performed in triplicate. Media and supplements were obtained from Gibco BRL (Carlsbad, CA, USA). PGE2 incubation
HPDL cells were seeded in 12-well plates at a density of 37,500 cells/cm2 and incubated for 24 h. Cells were starved in serum-free medium for 8 h and then the medium was replaced with either growth or osteogenic medium. The latter medium consisted of growth medium supplemented with 50 lg/mL of ascorbic acid, 250 nM dexamethasone and 5 mM beta-glycerophosphate (Sigma-Aldrich Chemical, St Louis, MO, USA). Cells were cultured with 0.001–1 lM prostaglandin E2 (Cayman Chemical, Ann Arbor, MI, USA), 0.001–1 lM butaprost, an EP2 agonist (Cayman Chemical), 0.6–42 nM sulprostone, an EP1 and EP3 agonist (Tocris Bioscience, Ellisville, MO, USA) or 1.25–2.5 nM TCS2510, an EP4 agonist (Tocris Bioscience).
PGE2 suppresses in-vitro calcification in HPDL cells For the protein kinase A (PKA) signaling pathway experiments, cells were cultured with 20 lM of a PKA inhibitor (adenosine 3′,5′-cyclic monophosphorothioate, Rp-Isomer, triethylammonium salt; Calbiochem, EMD Biosciences, Inc., San Diego, CA, USA) for 30 min before stimulation with PGE2. After culture for 24 h, total RNA was extracted and subjected to quantitative real-time PCR (qPCR; see below). Cellular protein levels were determined by western blot analysis. The most effective dose of PGE2 was selected and used for the rest of the experiments. Transfection of small interfering RNA
HPDL cells were grown in six-well plates and cultured in medium, without antibiotics, to 70–80% confluence. Cells were then treated, according to the manufacturer’s instructions, with a solution of small interfering (si) RNA oligonucleotides specific to EP2 (Santa Cruz Biotechnology, Dallas, TX, USA). Control siRNA (Santa Cruz Biotechnology) was used as a control treatment. Transfection with the siRNA was performed 24 h before exposure to PGE2. After 24 h of treatment with PGE2, mRNA was isolated for further analysis. Osteogenic differentiation
Cells were seeded at the same density, as described above, in a 24-well plate and were maintained in osteogenic medium. The medium was changed
every 48 h. Osteoblast marker gene expression and mineral deposition was investigated using the methods described below. Quantitative real-time PCR analysis
Total RNA was extracted using IsolRNA Lysis Reagent (5Prime, Gaitherburg, MD, USA) according to the manufacturer’s instructions. One microgram of each RNA sample was converted to cDNA by avian myeloblastosis virus reverse transcriptase (Promega, Madison, WI, USA) for 1.5 h at 42°C. Subsequently, qPCR was performed in a Lightcycler Nano real-time PCR system (Roche Applied Science, Indianapolis, IN, USA) using the SYBR Green system (Roche Applied Science). Amplification was performed as follows: 40 cycles of denaturation at 94°C for 20 s, annealing at 60°C for 20 s and extension at 72°C for 20 s. The reaction product was quantified with glyceraldehyde3-phosphate dehydrogenase (GAPDH) as the reference gene. The primers were designed based on the sequences reported in GenBank and are shown in Table 1. Mineralization assay
The cells were fixed with cold methanol for 10 min, washed with deionized water and stained with 1% Alizarin Red S solution for 3 min at room temperature. To quantify the amount of calcium deposited, the stain was eluted with 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate at room temperature for
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15 min and the absorbance was read at 570 nm. Protein extraction and western blot analysis
Protein was extracted with radioimmunoprecipitation buffer (50 mM Tris/ HCl, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% Na-deoxycholate) containing a cocktail of protease inhibitors and phosphatase inhibitors (1 mM sodium vanadate, 50 mM NaF; SigmaAldrich Chemical). Protein concentrations were measured using a BCA protein assay kit (Pierce Biotechnology, Rockford, IL, USA), and equal amounts of protein samples were electrophoresed through a 12% sodium dodecyl sulfate–polyacrylamide gel and subsequently transferred onto a nitrocellulose membrane. The membrane was incubated with primary antibody against TWIST1 (dilution 1 : 250; Abcam, Cambridge, UK), RUNX2 (dilution 1 : 1000; R & D Systems, Minneapolis, MN, USA) or GAPDH (dilution 1 : 2000; Chemicon International, Temecula, CA, USA) at 4°C. The membranes were then incubated with biotinylated secondary antibody, followed by peroxidase-labeled streptavidin. The signal was detected by chemiluminescence (Pierce Biotechnology) and quantified using an image analyzer (Vilber Lourmat, Marne-la-Vallee, France). Statistical analysis
All data were analyzed by one-way ANOVA using statistical software (SPSS, Chicago, IL, USA). Scheffe’s
Table 1. Primer sequences used for quantitative real-time PCR analysis
Gene symbol
Forward (5′–3′)
Reverse (5′–3′)
Accession number
RUNX2 (OSF2/ CBFA1a or RUNX2 type II) OCN TWIST1 TWIST2 GAPDH
ATG ATG ACA CTG CCA CCT CTG A
GGC TGG ATA GTG CAT TCG TG
NM_057179.2
CTT TGT GTC CAA GCA GGA GG TCT TAC GAG GAG CTG CAG ACG CA GCT GCG CAA GAT CAT CCC TCA TGG GTG TGA ACC ATG AGA A
CTG AAA GCC GAT GTG GTC AG ATC TTG GAG TCC AGC TCG TCG CT GTA GCT GCA GCT GGT CAT C GGC ATG GAC TGT GGT CAT GAG
NM_199173.4 NM_000474.3 NM_033012.2 NM_002046.3
GAPDH, glyceraldehyde-3-phosphate dehydrogenase; OCN, osteocalcin; RUNX2, Runt-related transcription factor 2; TWIST1, TWISTrelated protein1; TWIST2, TWIST-related protein2.
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test was used for post-hoc analysis ( p < 0.05).
Results In the first experiment, HPDL cells were cultured in osteogenic medium with or without PGE2 ranging in concentration from 0.001 to 1 lM. None of the concentrations of PGE2 used were toxic to the cells after 3 d in culture, as indicated by the MTT assay (data not shown). Osteogenic differentiation was determined based on in-vitro mineral deposition using Alizarin Red S staining. The results in Fig. 1 indicate that the addition of exogenous PGE2 to the cultures suppressed the in-vitro mineral deposition by HPDL cells in a dose-dependent manner. The amount of calcium deposited was quantified by eluting the stain with 10% cetylpyridinium chloride monohydrate and assessing the absorbance at 570 nm (Fig. 1). The quantitative results supported the decrease in calcium staining and a
significant inhibitory effect was found at concentrations of 0.01 lM PGE2 and higher. Our preliminary results indicated that the peak expression of mRNA for osteogenic markers in HPDL cells cultured in osteogenic medium occurred at day 10 (data not shown). Therefore, this time point was chosen to examine further the influence of PGE2 on osteogenesis in HPDL cells (Fig. 2). Interestingly, a dose-dependent upregulation of RUNX2 by PGE2 was observed, despite the inhibitory effect of PGE2 on mineral deposition. The expression of osteocalcin, a RUNX2 target gene and a marker of osteogenic differentiation, did not significantly change when exposed to PGE2. In addition, the results revealed increased expression of TWIST1, an inhibitor of RUNX2, but no effect on the expression of TWIST2, demonstrating that PGE2 induced TWIST1 expression. The inductive effect of PGE2 on TWIST1 expression was apparent at 0.01 lM PGE2 or higher.
Fig. 1. Prostaglandin E2 (PGE2) inhibits the osteogenic differentiation of human periodontal ligament (HPDL) cells in vitro. HPDL cells were cultured in osteogenic medium in the presence of 0.001–1 lM PGE2 for 18 d (D18). In-vitro mineral deposition was determined by Alizarin Red S staining. The bar chart shows the staining quantified by eluting the stain with 10% cetylpyridinium chloride monohydrate in 10 mM sodium phosphate at room temperature for 15 min followed by reading the absorbance at 570 nm. The staining at day 0 (D0) was used as background. The results represent the average + SD from three experiments. Asterisks indicate statistical significance at p < 0.05. The experiments were performed in triplicate using HPDL cell lines from three donors.
Next, we tested the short-term influence of PGE2 on HPDL cells to determine the direct influence of PGE2 on osteogenic-related gene expression. The results shown in Fig. 3 indicate that PGE2 increased the expression of RUNX2 and TWIST1 mRNAs within the first 24 h. In addition, analysis of the levels of RUNX2 and TWIST1 proteins was performed at 1, 2, 3 and 4 d (data not shown). However, an obvious increase was noted only at day 4 of treatment with PGE2 (Fig. 3). HPDL cells expressed all known EP receptors (Figure S1). To investigate the possible role of the EP receptors, cells were treated with EP agonists and the expression of RUNX2 and TWIST1 was examined by qPCR (Fig. 4A). Among the EP agonists used, only the EP2 agonist significantly increased the expression of RUNX2 and TWIST1. Furthermore, the increased expression of RUNX2 and TWIST1 induced by the EP2 agonist was found to be dosedependent, as shown in Fig. 4B. Gene silencing using siRNA was performed to analyze the possible role of the EP2 receptor. Transfection with siEP2 inhibited the expression of EP2 by more than 70% (Figure S2). The results from Fig. 4C reveal that the inductive effect of PGE2 on RUNX2 and TWIST1 expression was attenuated in HPDL cells transfected with siEP2. Because the EP receptor is associated with the cAMP–PKA signaling pathway, the involvement of PKA in the inductive effect of PGE2 was investigated. Cells were cultured with PGE2 in the presence or absence of a PKA inhibitor. The results indicated that the PKA inhibitor attenuates the inductive effect of PGE2 on TWIST1 expression (Fig. 5A). In addition, the application of forskolin, a PKA activator, up-regulated TWIST1 in a dose-dependent manner (Fig. 5B), in contrast to the effect of the PKA inhibitor.
Discussion The present study shows, for the first time, that PGE2 can modulate the
PGE2 suppresses in-vitro calcification in HPDL cells
Fig. 2. Prostaglandin E2 (PGE2) induces Runt-related transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1) expression by human periodontal ligament (HPDL) cells. Cells were treated with 0.001–1 lM PGE2 for 10 d. The expression of mRNAs for RUNX2, osteocalcin (OCN) and TWIST1 and TWIST-related protein2 (TWIST2) was analyzed by real-time PCR and the results are shown as relative expression compared with the control culture without PGE2. The level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control. The results represent the average + SD from three experiments. Asterisks indicate statistical significance at p < 0.05. The experiments were performed in triplicate using HPDL cell lines from three donors.
A
B
C
Fig. 3. Prostaglandin E2 (PGE2) increases both protein and mRNA expression of Runtrelated transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1). The cells were treated with 0.001–1 lM PGE2 for 1 d. The expression of mRNA for RUNX2 (A) and for TWIST1 and TWIST-related protein2 (TWIST2) (B) was detected by real-time PCR and the results are shown as relative expression compared with the control culture without PGE2, with the level of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) used as the internal control. The results represent the average + SD from three experiments. Asterisks indicate statistical significance at p < 0.05. Western blot analysis was performed to confirm the result from PCR analysis (C). The experiments were performed in triplicate using human periodontal ligament (HPDL) cell lines from three donors.
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expression of both RUNX2 and TWIST1, two key genes involved in the regulation of osteogenic differentiation, in HPDL cells. Despite inducing an increase in the expression of RUNX2 mRNA and RUNX2 protein, PGE2 inhibited in-vitro mineral deposition by HPDL cells cultured in osteogenic medium. This inhibitory effect might be caused by the upregulation of TWIST1, an inhibitor of RUNX2. The inductive effect of PGE2 on both genes was found to depend on the EP2–PKA signaling pathway. Periodontal ligament cells reside within the periodontal ligament that functions as a cushion to absorb the masticatory forces exerted upon the tooth. These cells possess several osteoblastic characteristics, such as the expression of alkaline phosphatase and the ability to generate in-vitro mineral deposition. We previously reported that periodontal ligament cells responded to applied pressure by releasing PGE2 (32,33). The results of the present study suggest a role for PGE2 in the homeostasis of the periodontal ligament. PGE2 might help to maintain the fibroblastic phenotype and prevent or inhibit the osteogenic differentiation of HPDL cells. PGE2 has been shown to exert both anabolic and catabolic effects on bone formation. The inductive effect of PGE2 on osteoclast formation has been reported (34). PGE2 is a potent inducer of RANKL, which plays an essential role in osteoclastogenesis via its interaction with RANK on the osteoclast precursor surface. Moreover, it was reported that PGE2 may play a role in bone formation (17). PGE2 has been shown to promote the proliferation and differentiation of marrow stromal cells in vitro (14) and induced cancellous bone formation in rats (17). However, in contrast to HPDL cells, PGE2 induced RUNX2 expression and in-vitro mineral deposition by rat marrow stromal cells (21). The difference in the results is probably caused by the different cell types and species used in these studies. Our previous results also indicated different responses between HPDL cells and mouse marrow stromal cells (35). In that study, capsaicin induced osteoprotegerin expression
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A
B
C
Fig. 4. E prostanoid receptor 2 (EP2) involvement in prostaglandin E2 (PGE2)-induced Runt-related transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1) expression. Cells were treated with EP1–4 agonists and the expression of RUNX2 and TWIST1 were determined by real-time PCR (A). Various doses of EP2 agonist were used to further confirm the effect on expression of RUNX2 and TWIST1 mRNAs (B). EP2 silencing using small interfering RNA (siRNA) abolished the inductive effect of PGE2 on RUNX2 and TWIST1 expression (C). The results are shown as relative expression from three experiments using human periodontal ligament (HPDL) cells from three donors.
in HPDL cells, whereas it induced RANKL expression in mouse marrow stromal cells. The exact mechanisms underlying these differences are still unclear. PGE2 exerts its effect via EP receptors. Among the four subtypes of EP receptor, EP2 and EP4 are the main receptors that participate in mineralized tissue homeostasis. The application of EP2 and EP4 agonists has been demonstrated to either induce or inhibit bone formation (21,36–39). Therefore, the exact role of each of these two receptors remains unclear. It has been reported that PGE2 induces RUNX2 expression and mineralization via EP2 and EP4 in rat and mouse bone marrow cultures (21,39). In addition, EP2 and EP4 agonists increased bone formation in an
animal model (21,36). EP2 and EP4 have also been shown to participate in PGE2-induced bone resorption (37,38,40). The mechanisms underlying the anabolic or catabolic effects of EP2 and EP4 in bone homeostasis are as yet unknown. It is possible that EP receptors have functional redundancy and that the chemical agonist used in those experiments might activate more than one EP receptor. In the present study, the EP4 agonist did not have any effect on the expression of RUNX2 or TWIST1. The application of the EP2 agonist, however, showed an effect similar to that seen with PGE2. The role of EP2 was confirmed by the use of a silencing approach, indicating the importance of EP2 in HPDL cells in their response to PGE2.
It has been demonstrated that the EP2 receptor activated by PGE2 resulted in an increased level of cytosolic cAMP (41,42). Subsequent to its formation, cAMP can either activate adenylate kinase or translocate into the nucleus. In the present study, PKA inhibition reduced the expression of RUNX2 and TWIST1, suggesting involvement of the EP2–PKA pathway. The role of EP2–PKA was reported for different biological processes, such as mast cell degranulation or interleukin-8 expression by colonic epithelial cells (41,42). Whether this pathway is involved in RUNX2 and TWIST1 expression in other cell types needs further investigation. RUNX2 (or CBFA1) is a key regulator of osteogenic differentiation (22,23). In RUNX2 knockout mice, mineralized bone is absent because of a defect in osteoblast differentiation (22). Our finding, that PGE2 upregulates RUNX2, suggests that the anabolic effect of PGE2 might occur via this induction. Interestingly, we found that, despite increased expression of RUNX2, in-vitro mineral deposition was inhibited. It is possible that PGE2 exerted this inhibitory effect via the upregulation of TWIST1 because TWIST1 has been demonstrated to be able to bind to RUNX2 and inhibit its function. Although TWIST inhibited the function of RUNX2, it had no effect on RUNX2 expression (24). Moreover, in TWIST1+/ mice, premature osteoblast differentiation and premature trabecular bone formation was detected in embryonic day 16 embryos (24). The inhibitory role of TWIST1 coincides with the observation that the expression of osteocalcin was not increased. As osteocalcin is one of the target molecules of RUNX2, the explanation could be that TWIST1 binds to RUNX2 and thereby prevents the function of this transcription factor. In conclusion, PGE2 was shown to regulate the expression of RUNX2 and TWIST1 mRNAs and RUNX2 and TWIST1 proteins. Our data strongly suggest the involvement of the EP2–PKA signaling pathway in this process. The balance between RUNX2 and TWIST1 may regulate
PGE2 suppresses in-vitro calcification in HPDL cells A
B
7.
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9.
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Fig. 5. The inductive effect of prostaglandin E2 (PGE2) occurs via the protein kinase A (PKA) pathway. Cells were treated with 1 lM PGE2, and the expression of Runt-related transcription factor 2 (RUNX2) and TWIST-related protein1 (TWIST1) was determined by real-time PCR. Application of a PKA inhibitor (PKAi) inhibited the inductive effect of PGE2. Moreover, when cells were treated with forskolin, a PKA activator, upregulation of RUNX2 and TWIST1 was observed.
the differentiation state of PDL cells. The level and duration of exposure to PGE2 may help to regulate the behavior of PDL cells and help to modulate the homeostasis of periodontal tissue.
Acknowledgements This work was supported by “Integrated Innovation Academic Center: IIAC” Chulalongkorn University Centenary Academic Development Project (CU05_55_32) and the Research Chair Grant of the National Science and Technology Development Agency.
Source of funding Integrated Innovation Academic Center (IIAC), Chulalongkorn University Centenary Academic Development Project; The National Science and Technology Development Agency (NSTDA) Research Chair Grant.
Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Human PDL cells express EP receptors.
Figure S2 siRNA reduced EP2 expression.
application
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References 1. Akaogi J, Nozaki T, Satoh M, Yamada H. Role of PGE2 and EP receptors in the pathogenesis of rheumatoid arthritis and as a novel therapeutic strategy. Endocr Metab Immune Disord Drug Targets 2006;6:383–394. 2. Kalinski P. Regulation of immune responses by prostaglandin E2. J Immunol 2012;188:21–28. 3. Aronoff DM, Canetti C, Peters-Golden M. Prostaglandin E2 inhibits alveolar macrophage phagocytosis through an E-prostanoid 2 receptor-mediated increase in intracellular cyclic AMP. J Immunol 2004;173:559–565. 4. Huang S, Wettlaufer SH, Hogaboam C, Aronoff DM, Peters-Golden M. Prostaglandin E(2) inhibits collagen expression and proliferation in patient-derived normal lung fibroblasts via E prostanoid 2 receptor and cAMP signaling. Am J Physiol Lung Cell Mol Physiol 2007;292:L405–L413. 5. Kolodsick JE, Peters-Golden M, Larios J, Toews GB, Thannickal VJ, Moore BB. Prostaglandin E2 inhibits fibroblast to myofibroblast transition via E. prostanoid receptor 2 signaling and cyclic adenosine monophosphate elevation. Am J Respir Cell Mol Biol 2003;29:537–544. 6. White ES, Atrasz RG, Dickie EG et al. Prostaglandin E(2) inhibits fibroblast
13.
14.
15.
16.
17.
18.
783
migration by E-prostanoid 2 receptormediated increase in PTEN activity. Am J Respir Cell Mol Biol 2005;32:135–141. Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–342. Coetzee M, Haag M, Kruger MC. Effects of arachidonic acid and docosahexaenoic acid on differentiation and mineralization of MC3T3-E1 osteoblastlike cells. Cell Biochem Funct 2009;27: 3–11. Hagino H, Kuraoka M, Kameyama Y, Okano T, Teshima R. Effect of a selective agonist for prostaglandin E receptor subtype EP4 (ONO-4819) on the cortical bone response to mechanical loading. Bone 2005;36:444–453. Minamizaki T, Yoshiko Y, Kozai K, Aubin JE, Maeda N. EP2 and EP4 receptors differentially mediate MAPK pathways underlying anabolic actions of prostaglandin E2 on bone formation in rat calvaria cell cultures. Bone 2009;44: 1177–1185. Breyer RM, Bagdassarian CK, Myers SA, Breyer MD. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol 2001;41:661–690. Alander CB, Raisz LG. Effects of selective prostaglandins E2 receptor agonists on cultured calvarial murine osteoblastic cells. Prostaglandins Other Lipid Mediat 2006;81:178–183. Paralkar VM, Borovecki F, Ke HZ et al. An EP2 receptor-selective prostaglandin E2 agonist induces bone healing. Proc Natl Acad Sci U S A 2003;100: 6736–6740. Weinreb M, Shamir D, Machwate M, Rodan GA, Harada S, Keila S. Prostaglandin E2 (PGE2) increases the number of rat bone marrow osteogenic stromal cells (BMSC) via binding the EP4 receptor, activating sphingosine kinase and inhibiting caspase activity. Prostaglandins Leukot Essent Fatty Acids 2006;75:81–90. Raisz LG, Woodiel FN. Effects of selective prostaglandin EP2 and EP4 receptor agonists on bone resorption and formation in fetal rat organ cultures. Prostaglandins Other Lipid Mediat 2003;71: 287–292. Zhang J, Wang JH. BMP-2 mediates PGE(2) -induced reduction of proliferation and osteogenic differentiation of human tendon stem cells. J Orthop Res 2012;30:47–52. Keila S, Kelner A, Weinreb M. Systemic prostaglandin E2 increases cancellous bone formation and mass in aging rats and stimulates their bone marrow osteogenic capacity in vivo and in vitro. J Endocrinol 2001;168:131–139. Ke HZ, Crawford DT, Qi H et al. A nonprostanoid EP4 receptor selective
784
19.
20.
21.
22.
23.
24.
25.
26.
27.
Manokawinchoke et al.
prostaglandin E2 agonist restores bone mass and strength in aged, ovariectomized rats. J Bone Miner Res 2006;21:565–575. Li M, Thompson DD, Paralkar VM. Prostaglandin E(2) receptors in bone formation. Int Orthop 2007;31:767–772. Tanaka M, Sakai A, Uchida S et al. Prostaglandin E2 receptor (EP4) selective agonist (ONO-4819.CD) accelerates bone repair of femoral cortex after drill-hole injury associated with local upregulation of bone turnover in mature rats. Bone 2004;34:940–948. Yoshida K, Oida H, Kobayashi T et al. Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation. Proc Natl Acad Sci U S A 2002;99:4580–4585. Komori T, Yagi H, Nomura S et al. Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 1997;89:755–764. Otto F, Thornell AP, Crompton T et al. Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 1997;89:765–771. Bialek P, Kern B, Yang X et al. A twist code determines the onset of osteoblast differentiation. Dev Cell 2004;6:423–435. Yousfi M, Lasmoles F, Marie PJ. TWIST inactivation reduces CBFA1/ RUNX2 expression and DNA binding to the osteocalcin promoter in osteoblasts. Biochem Biophys Res Commun 2002;297:641–644. el Ghouzzi V, Le Merrer M, PerrinSchmitt F et al. Mutations of the TWIST gene in the Saethre-Chotzen syndrome. Nat Genet 1997;15:42–46. Howard TD, Paznekas WA, Green ED et al. Mutations in TWIST, a basic helixloop-helix transcription factor, in
28.
29.
30.
31.
32.
33.
34.
Saethre-Chotzen syndrome. Nat Genet 1997;15:36–41. Yousfi M, Lasmoles F, Lomri A, Delannoy P, Marie PJ. Increased bone formation and decreased osteocalcin expression induced by reduced Twist dosage in Saethre-Chotzen syndrome. J Clin Investig 2001;107:1153–1161. Miraoui H, Severe N, Vaudin P, Pages JC, Marie PJ. Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells: implication of FGFR2 signaling. J Cell Biochem 2010;110:1147–1154. Lee MS, Lowe GN, Strong DD, Wergedal JE, Glackin CA. TWIST, a basic helix-loop-helix transcription factor, can regulate the human osteogenic lineage. J Cell Biochem 1999;75:566–577. Danciu TE, Li Y, Koh A, Xiao G, McCauley LK, Franceschi RT. The basic helix loop helix transcription factor Twist1 is a novel regulator of ATF4 in osteoblasts. J Cell Biochem 2012;113: 70–79. Luckprom P, Wongkhantee S, Yongchaitrakul T, Pavasant P. Adenosine triphosphate stimulates RANKL expression through P2Y1 receptor-cyclo-oxygenasedependent pathway in human periodontal ligament cells. J Periodontal Res 2010;45:404–411. Wongkhantee S, Yongchaitrakul T, Pavasant P. Mechanical stress induces osteopontin expression in human periodontal ligament cells through rho kinase. J Periodontol 2007;78:1113–1119. Kanzaki H, Chiba M, Shimizu Y, Mitani H. Periodontal ligament cells under mechanical stress induce osteoclastogenesis by receptor activator of nuclear factor kappaB ligand up-regulation via prostaglandin E2 synthesis. J Bone Miner Res 2002;17:210–220.
35. Sooampon S, Manokawinchoke J, Pavasant P. Transient receptor potential vanilloid-1 regulates osteoprotegerin/ RANKL homeostasis in human periodontal ligament cells. J Periodontal Res 2013;48:22–29. 36. Kambe T, Maruyama T, Nakai Y et al. Discovery of novel prostaglandin analogs as potent and selective EP2/EP4 dual agonists. Bioorg Med Chem 2012;20: 2235–2251. 37. Sakuma Y, Tanaka K, Suda M et al. Crucial involvement of the EP4 subtype of prostaglandin E receptor in osteoclast formation by proinflammatory cytokines and lipopolysaccharide. J Bone Miner Res 2000;15:218–227. 38. Suzawa T, Miyaura C, Inada M et al. The role of prostaglandin E receptor subtypes (EP1, EP2, EP3, and EP4) in bone resorption: an analysis using specific agonists for the respective EPs. Endocrinology 2000;141:1554–1559. 39. Weinreb M, Grosskopf A, Shir N. The anabolic effect of PGE2 in rat bone marrow cultures is mediated via the EP4 receptor subtype. Am J Physiol 1999;276: E376–E383. 40. Li X, Okada Y, Pilbeam CC et al. Knockout of the murine prostaglandin EP2 receptor impairs osteoclastogenesis in vitro. Endocrinology 2000;141:2054–2061. 41. Serra-Pages M, Olivera A, Torres R, Picado C, de Mora F, Rivera J. E-prostanoid 2 receptors dampen mast cell degranulation via cAMP/PKA-mediated suppression of IgE-dependent signaling. J Leukoc Biol 2012;92:1155–1165. 42. Srivastava V, Dey I, Leung P, Chadee K. Prostaglandin E2 modulates IL-8 expression through formation of a multiprotein enhanceosome in human colonic epithelial cells. Eur J Immunol 2012;42:912–923.
