Elsevier

Archives of Oral Biology

Volume 57, Issue 9, September 2012, Pages 1274-1281
Archives of Oral Biology

Prostaglandin E2 affects osteoblast biology in a dose-dependent manner: An in vitro study

https://doi.org/10.1016/j.archoralbio.2012.03.003Get rights and content

Abstract

Objective

This study aimed to determine in vitro how exogenous PGE2 affects the expression of genes in cultured osteoblasts by relative quantitation PCR.

Design

Cultured osteoblasts were exposed to 10−3 M, 10−5 M or 10−7 M PGE2 over 5, 10, 15 and 20 days.

Results

RANKL expression was higher after 5 days of exposure (p < 0.05), but thereafter reduced in those treated with the two lower doses of PGE2 (p < 0.01). RANKL/OPG ratio reported in favour of OPG gene expression and alkaline phosphatase gene expression increased in osteoblasts exposed to the two lower doses of the eicosanoid after 15 days. Conversely, prostaglandin E synthase, a cytokine produced during PGE2 synthesis, gene expression was significantly reduced at 15 and 20 days (p < 0.01 and 0.05 respectively). The results from this study add to the current knowledge of the mechanisms by which PGE2 modulates the osteoblast biology in a dose-dependent manner.

Conclusions

It is proposed that PGE2at a low dose switch osteoblast's biology in favour of bone apposition by: first, inducing a significantly higher OPG gene expression overwhelming RANKL gene expression; second, reducing PGEs synthesis; and third, increasing ALP gene expression. An opposite effect is expected when the concentration of the eicosanoid overpass certain levels.

Introduction

Prostaglandin E2 (PGE2) at low doses released locally at the mandible has been reported to stimulate higher rates of bone deposition,1 which associates to an increase in TGF-β1 and TGF-β receptors synthesis, as well as a reduction in the number of osteoclasts resorbing alveolar bone.1, 2, 3 Those reports confirmed that in rats exogenous PGE2 delivered in a low dose (≤0.5 μg) stimulates bone apposition, whereas higher doses result in bone resorption. Although reports from these authors have partially explained the effects produced by PGE2 in a dose-dependent manner on the bone cells,2, 3 up today other mechanisms by which this eicosanoid can affect the bone cells are unknown.

The nuclear factor Kβ-ligand (RANKL), also known as tumour necrosis factor-related activated induced cytokine (TRANCE), osteoclast differentiation factor (ODF), or osteoprotegerin ligand (OPGL), is synthesized by osteoblasts and expressed as a decoy receptor in the osteoblast's membrane.4 By cell to cell contact, it binds a membrane protein (RANK) expressed by monocyte/macrophage cells and enables differentiation of monocytes/macrophages into osteoclasts.5, 6 Another cytokine synthesized by the osteoblast, osteoprotegerin (OPG), also known as osteoclast inhibitory factor (OIF), is released into the extracellular media where it competes to bind RANKL. OPG bound to RANKL does not permit the cell to cell contact during osteoclast differentiation, inhibiting osteoclast differentiation.7, 8

Bone matrix is deposited by the osteoblasts during the stages of differentiation and maturation.9 Alkaline phosphatase (ALP) is synthesized by osteoblasts and acts as an initiator of bone matrix biomineralization.10, 11 During osteoblast differentiation and maturation, prior to the increase in ALP expression, there is an increase in PGE2 synthesis.12 An increase in ALP activity in osteoblasts is followed by a decrease in PGE2 synthesis.13

An in vitro study was designed to determine how exogenous PGE2 at different concentrations, affect the gene expression of RANKL, OPG, ALP and PGE synthase (PGEs), a cytokine produced during PGE2 synthesis. Thus, the specific aims of this study were first, to clarify how exogenous PGE2 affects the gene expression of RANKL and OPG in cultured primary osteoblasts; second, to elucidate variations in RANKL/OPG gene expression ratio in cultured osteoblasts when exposed to various concentrations of PGE2; and third, to determine how the eicosanoid affects ALP and PGEs gene expression in cultured osteoblasts.

Section snippets

Material and methods

Primary osteoblasts from the head and long bones of 3 Wistar male rats, 8 weeks old, were harvested, manually defleshed and cultured as described previously.2 After reaching 70–80% confluence, approximately 2 weeks of culture, cells were treated with 3 ml of 0.25% Trypsin (Gibco, Cat. No. 07476, USA) at 37 °C for 3 min and plated at 141 × 103 (±28.5) cells/ml in 75 ml flasks containing 3 ml of the supplemented DMEM as described above. When the cultured cells again reached 80–90% confluence, they were

Results

The most of the cultured cells showed a cuboidal shape and immunoreactivity for Cbfa-1. Alkaline phosphatase activity was observed in all the cells tested for this cytokine. All the experiments were run on passage two.

Discussion

This in vitro study has shown that PGE2 has an effect on osteoblast biology by either stimulating or inhibiting RANKL, OPG, ALP and PGEs gene expression conditional to time and concentration used. Thus, these results add to previous reports,2, 3 by further explaining how PGE2 affects in a dose-dependent manner the bone remodelling cycle.1

A dose dependent effect of the eicosanoid on bone cells has been previously reported by others.16, 17, 18 The OPG/RANKL ratios computed in this study at

Conclusions

This in vitro study further explains the results reported in previous in vivo studies and produces insights in some other pathways by which PGE2 can modulate the bone remodelling cycle in a dose-dependent manner. The current results demonstrated that exogenous PGE2 at a lower concentration can modulate osteoblasts biology by: first, inducing a significantly higher OPG gene expression overwhelming RANKL gene expression in the osteoblasts; second, reducing PGEs synthesis in the osteoblasts; and

Funding

This study was supported by the National Health Research Council of Australia.

Competing interests

The authors declare that no conflict of interest or any financial or personal relationship exists between them and the manufacturers of the products used in this study.

Ethical approval

The presented study was approved by the University of Queensland, Animal Ethics Committee (DENT/046/02).

Acknowledgements

The authors want to acknowledge Dr. Bob Simpson from the Biochemistry School at The University of Queensland for his technical support with the real time polymerase chain reaction. Also, thanks to the National Health Research Council of Australia for its financial support for this study.

References (22)

  • T. Suda et al.

    The molecular mechanism of osteoclastogenesis: ODF/RANKL-dependent and independent pathways

  • Cited by (18)

    • Synthesis, swelling, degradation and cytocompatibility of crosslinked PLLA-PEG-PLLA networks with short PLLA blocks

      2016, European Polymer Journal
      Citation Excerpt :

      To establish a confluent monolayer of primary osteoblasts, tissue was placed in the MEM culture media (GIBCO, Invitrogen, USA; supplemented with 20% (v/v) fetal calf serum (FCS), Morgate Biotech, Australia; 2% penicillin (10,000 U mL−1) and streptomycin, 10,000 μg mL−1 (PNS), GIBCO, Invitrogen, USA and 0.2% Fungizone® Antimycotic liquid (1 μg mL−1), GIBCO, Invitrogen, USA) at 37 ± 0.5 °C, pH 7.4 with 5% CO2/95% atmospheric air for 7 days [25]. The primary osteoblasts established in a 6-well tissue culture plate (TPP Techno Plastic Products, Switzerland) were sub-cultured until sufficient cell numbers were available for the biocompatibility tests of hydrogels on cultured osteoblasts [26–28]. Cells were characterized as osteoblasts by their morphology (cuboidal shaped) when examined under inverted light microscope [27].

    • Prostaglandin E<inf>2</inf> impairs osteogenic and facilitates adipogenic differentiation of human bone marrow stromal cells

      2015, Prostaglandins Leukotrienes and Essential Fatty Acids
      Citation Excerpt :

      Prostaglandin E2 (PGE2) has a substantial influence on bone remodeling and bone healing, but its effects are still controversially discussed [7]. Whereas low concentrations of PGE2 are considered to have an anabolic effect, high concentrations of PGE2 lead preferentially to osteoclast activation and contribute to inflammatory bone lost/destruction [15]. Also the time point and duration of exposition to PGE2 strongly determines its effect on bone remodeling.

    • NSAIDs can have adverse effects on bone healing

      2013, Medical Hypotheses
      Citation Excerpt :

      They can cause the development of osteoblasts, but also cause precursor cells of osteoblasts to control bone resorption. Additionally, PGs can both induce and inhibit the formation and growth of osteoblasts [8,9]. Cyclo-oxygenases (COX) play an important role in the production of PGs. These enzymes in bone tissue may show increased activity under the influence of Hypoxia Inducible Factor, which is released when oxygen concentrations are low.

    • Osteogenic differentiation of periodontal fibroblasts is dependent on the strength of mechanical strain

      2013, Archives of Oral Biology
      Citation Excerpt :

      They concluded that high strain was pro-inflammatory and enhanced the IL1β-stimulated synthesis of inflammatory molecules, which in turn inhibited ALP activity. This might also be the reason for the reduced OPG synthesis by HOB at 10% of SMS, because it is known that inflammation cytokines can inhibit OPG synthesis.41 The results for OPG and ALP show discrepancies between the results on the mRNA and protein level.

    • Ketorolac and bone healing: a review of the basic science and clinical literature

      2024, European Journal of Orthopaedic Surgery and Traumatology
    View all citing articles on Scopus
    View full text