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. 2009 Feb;44(2):266-74.
doi: 10.1016/j.bone.2008.10.041. Epub 2008 Oct 25.

In vitro and in vivo evidence for stimulation of bone resorption by an EP4 receptor agonist and basic fibroblast growth factor: Implications for their efficacy as bone anabolic agents

M E Downey  1 , L S Holliday, J I Aguirre, T J Wronski
Affiliations

In vitro and in vivo evidence for stimulation of bone resorption by an EP4 receptor agonist and basic fibroblast growth factor: Implications for their efficacy as bone anabolic agents

M E Downey et al. Bone. 2009 Feb.

Abstract

Prostaglandin E2 receptor subtype 4 agonists (EP4A) and basic fibroblast growth factor (FGF2) stimulate bone formation, but their effects on bone resorption are controversial. To provide additional insight into the skeletal effects of EP4A and FGF2, their regulation of expression of genes associated with bone formation and resorption in aged ovariectomized (OVX) rats and in cultured mouse bone marrow cells was determined. RNA was isolated from lumbar vertebrae of OVX rats (16 months of age) treated daily for 3 weeks with FGF2 or EP4A and processed for quantitative real time-PCR analyses. mRNA expression for the receptor activator of NF-kappaB ligand (RANKL) and cathepsin K (CTSK), but not osteoprotegerin (OPG), were upregulated by both FGF2 and EP4A. Addition of FGF2 and EP4A to the medium of cultured mouse bone marrow cells increased the formation of tartrate resistant acid phosphatase (TRAP) positive cells, upregulated the expression of RANKL and CTSK, and downregulated expression for OPG. EP4A also increased the formation of actin rings, an indicator of osteoclast activation, in a dose dependent manner in osteoclasts cultured on bone slices and triggered the formation of pits as revealed by a pitting assay. Gene expression for osterix (OSX) and IGF-2, genes associated with bone formation, was significantly greater in FGF2-treated OVX rats compared with EP4A-treated OVX rats. These findings at the molecular level are consistent with previous tissue-level histomorphometric findings, and at the doses tested, support the contention that FGF2 has a stronger bone anabolic effect than EP4A. The results of these in vivo and in vitro analyses clarify the effects of FGF2 and EP4A on bone formation and resorption, and provide insight into differences in the efficacy of two potential bone anabolic agents for restoration of lost bone mass in the osteopenic, estrogen-deplete skeleton.

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Figures

Figure 1
Figure 1
Cancellous bone tissue in the lumbar vertebral body of an aged OVX rat treated for 3 weeks with the EP4 agonist, which induced formation of large, multinucleated osteoclasts (arrow) within resorptive lacunae. EP4 treatment increased osteoclast surface, an index of bone resorption, by nearly three-fold compared with baseline OVX rats. (Von Kossa/tetrachrome stain; original magnification, X400).
Figure 2
Figure 2
Effects of FGF2 and EP4 on the expression of A. OSX, B. IGF-2, C. TGF-β1, in lumbar vertebral bodies from of the following groups: Baseline OVX (BSL OVX), and OVX rats treated for 3 weeks with FGF2 (OVX+FGF2) or EP4 (OVX+EP4). Bars indicate mean ± SD of 5 animals. aSignificantly different from the BSL OVX group (P<0.05). bSignificantly different from the OVX+FGF2 group (P<0.05).
Figure 3
Figure 3
Effects of FGF2 and EP4 on the expression of A. COX-2 and B. VEGF, in lumbar vertebral bodies from of the following groups: Baseline OVX (BSL OVX), and OVX rats treated for 3 weeks with FGF2 (OVX+FGF2) or EP4 (OVX+EP4). Bars indicate mean ± SD of 5 animals. aSignificantly different from the BSL OVX group (P<0.05).
Figure 4
Figure 4
FGF2 and EP4 regulate the expression of A. RANKL, B. OPG, and C. CTSK mRNAs associated with bone resorption in lumbar vertebral bodies from ovariectomized (OVX) rats. Figures present fold changes relative to r18s from of the following experimental groups: Baseline OVX (BSL OVX), and OVX rats treated for 3 weeks with FGF2 (OVX+FGF2) or EP4 (OVX+EP4). Values are the mean ± SD from 5 OVX rats per group. aSignificantly different from BSL OVX (P<0.05).
Figure 5
Figure 5
Effects of FGF2, EP4, and 1,25-dihydroxivitamin D3 (1,25D3) on osteoclast formation. Mouse bone marrow was cultured with FGF2, EP4, 1,25D3, FGF2 + 1,25D3 and EP4 + 1,25D3 for 7 days. TRAP-positive cells were identified as mononuclear (one nucleus), multinuclear (2–10 nuclei), or giant (>10 nuclei) cells. Values are expressed as the mean ± SD for triplicate cultures. aSignificantly different from control (untreated mouse bone marrow culture) (P<0.05). bSignificantly different from mouse bone marrow culture treated with 1,25D3 alone (P<0.05).
Figure 6
Figure 6
Effects of COX-2 inhibitor on osteoclast differentiation. Mouse bone marrow cells were cultured with EP4, PGE2, 1,25D3, or EP4 + 1,25D3 with and without COX-2 inhibitor (NS-398) for 7 days. TRAP-positive cells were identified as mononuclear (one nucleus), multinuclear (2–10 nuclei), or giant (>10 nuclei) cells. No TRAP+ giant cells were detected in the cultures treated with the COX-2 inhibitor alone. Values are expressed as the mean ± SD for triplicate cultures. aSignificantly different from control (COX-2 inhibitor-treated mouse bone marrow culture) (P<0.05). bSignificantly different from mouse bone marrow culture treated with EP4 + COX-2 inhibitor (P<0.05). CSignificantly different from mouse bone marrow culture treated with 1,25D3 + COX-2 inhibitor (P<0.05).
Figure 7
Figure 7
Effects of EP4 and 1,25D3 on the formation of actin rings and resorptive pits on bone slices. Marrow cells that had been cultured for 5 days with 1,25D3 were scraped and plated on bone slices. Pits were determined by examining bone slices using differential interference contrast optics. A. Cultures treated with 1,25D3 alone, B. Cultures treated with 60 nM EP4 + 1,25D3, C. Cultures treated with 600 nM EP4 +1,25D3 with arrows showing the actin rings. Scale bar = 25 μm, D. Quantitation of the actin rings showing that their formation in response to EP4 is dose dependent, E. Quantitation of the pitting is shown in the bar graph. Values are expressed as the mean ± SD of 5 measurements. aSignificantly different from culture treated with 1,25D3 (P<0.05).
Figure 8
Figure 8
Effects of FGF2 and EP4 on the expression of A. RANKL, B. RANK, C. OPG, and D. CTSK mRNA in mouse bone marrow cells. Cultures were treated with FGF2, EP4, 1,25D3, FGF2 + 1,25D3 or EP4 + 1,25D3. Figures represent the fold induction relative to the untreated bone marrow culture. aSignificantly different from control (untreated mouse bone marrow culture) (P<0.05). bSignificantly different from mouse bone marrow culture treated with 1,25D3 alone (P<0.05).
Figure 9
Figure 9
Effects of FGF2 and EP4 on gene expression for COX-2 in mouse bone marrow cells. Cultures were treated with FGF2, EP4, 1,25D3, FGF2 + 1,25D3 or EP4 + 1,25D3. Figures represent the fold induction relative to the untreated bone marrow culture (control). aSignificantly different from control (P<0.05).
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