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Published on: December 11, 2010
Effects of prostaglandin E2 on gene expression in primary osteoblastic cells from prostaglandin receptor knockout
X Li1, C C Pilbeam, L Pan
1Department of Medicine, University of Connecticut Health Center, Farmington, CT 06030, USA.
Abstract:
Recent studies have shown that stimulation of osteoclastogenesis in cocultures of osteoblasts and spleen cells in response to prostaglandin E2 (PGE2) is markedly decreased when the osteoblasts are derived from cells lacking either the EP2 or the EP4 receptor. Induction of osteoclast formation requires upregulation of receptor activator of nuclear factor-kappaB ligand (RANKL) on cells of the osteoblastic lineage, which then binds to the RANK receptor on cells of the osteoclast lineage. Osteoprotegerin (OPG) is a decoy receptor for RANKL that can block its interaction with RANK. In addition, macrophage-colony stimulating factor (M-CSF) is essential for osteoclast formation. Finally, PGE2 can increase interleukin-6 (IL-6), which may further enhance osteoclastogenesis. To study the relative influence of the EP2 and EP4 receptors on response of these factors to PGE2, we examined mRNA levels for RANKL, OPG, M-CSF, and IL-6 in primary osteoblastic cell cultures derived from two lines of EP2 knockout mice (EP2-/-) and one line of EP4 knockout mice (EP4-/-) and the relevant wild-type controls (EP2+/+ and EP4+/+). The responses of cells from wild-type animals of all three lines were similar. After PGE2 treatment, RANKL mRNA levels were increased at 2 h, and this was sustained over 72 h. Basal RANKL expression was moderately reduced in EP2-/- cells and markedly reduced in EP4-/- cells. PGE2 increased RANKL mRNA in EP2-/- cells and EP4-/- cells, but the levels were significantly reduced compared with wild-type cells. There were no consistent changes in expression of M-CSF or OPG in the different genotypes or with PGE2 treatment. IL-6 mRNA was variably increased by PGE2 in both wild-type and knockout cells, although the absolute levels were somewhat lower in both EP2-/- and EP4 -/- cultures. Parathyroid hormone (PTH) increased RANKL and IL-6 and decreased OPG mRNA levels similarly in both wild-type and EP2-/- or EP4-/- cells. The major defect in the response to PGE2 in animals lacking either EP2 or EP4 receptors is a reduction in basal and stimulated RANKL levels. Loss of EP4 receptor appears to have a greater effect on basal RANKL expression than EP2.
Insights
Prostaglandin E2 (PGE2) signaling through EP2 and EP4 receptors is crucial for osteoclastogenesis. Loss of these receptors, particularly EP4, significantly reduces receptor activator of nuclear factor-kappaB ligand (RANKL) expression, impacting bone formation.
Area of Science:
- Bone Biology and Endocrinology
- Cell Signaling and Receptor Function
Background:
- Osteoclastogenesis, the formation of bone-resorbing cells, is a critical process in bone remodeling.
- Prostaglandin E2 (PGE2) is known to stimulate osteoclastogenesis, involving key mediators like receptor activator of nuclear factor-kappaB ligand (RANKL).
- EP2 and EP4 receptors are implicated in mediating PGE2's effects on osteoblasts and subsequent osteoclast formation.
Purpose of the Study:
- To investigate the specific roles of EP2 and EP4 receptors in mediating PGE2-induced changes in osteoclastogenesis-related gene expression.
- To determine the relative contribution of EP2 and EP4 receptors to basal and stimulated RANKL, OPG, M-CSF, and IL-6 mRNA levels.
Main Methods:
- Primary osteoblastic cell cultures were established from EP2 and EP4 knockout mice (EP2-/-, EP4-/-) and their wild-type littermate controls (EP2+/+, EP4+/+).
- Cells were treated with PGE2, and mRNA levels for RANKL, OPG, M-CSF, and IL-6 were quantified using quantitative real-time PCR.
- Parathyroid hormone (PTH) was used as a control stimulus to assess receptor-independent signaling pathways.
Main Results:
- PGE2 treatment robustly increased RANKL mRNA in wild-type osteoblasts.
- Basal RANKL mRNA levels were reduced in EP2-/- cells and markedly reduced in EP4-/- cells.
- While PGE2 still increased RANKL in knockout cells, the levels were significantly lower compared to wild-type, with EP4 deficiency showing a greater impact on basal RANKL.
Conclusions:
- The EP2 and EP4 receptors play a significant role in mediating PGE2-induced RANKL expression in osteoblasts.
- Loss of EP4 receptor has a more pronounced effect on basal RANKL expression than EP2.
- These findings highlight the importance of EP2 and EP4 signaling in regulating osteoclastogenesis through RANKL modulation.
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