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