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. 2002 Apr 2;99(7):4580-5.
doi: 10.1073/pnas.062053399. Epub 2002 Mar 26.

Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation

Keiji Yoshida  1 , Hiroji Oida, Takuya Kobayashi, Takayuki Maruyama, Masaharu Tanaka, Teruaki Katayama, Kojiro Yamaguchi, Eri Segi, Tadao Tsuboyama, Mutsumi Matsushita, Kosei Ito, Yoshiaki Ito, Yukihiko Sugimoto, Fumitaka Ushikubi, Shuichi Ohuchida, Kigen Kondo, Takashi Nakamura, Shuh Narumiya
Affiliations

Stimulation of bone formation and prevention of bone loss by prostaglandin E EP4 receptor activation

Keiji Yoshida et al. Proc Natl Acad Sci U S A. .

Abstract

Bone remodeling, comprising resorption of existing bone and de novo bone formation, is required for the maintenance of a constant bone mass. Prostaglandin (PG)E2 promotes both bone resorption and bone formation. By infusing PGE2 to mice lacking each of four PGE receptor (EP) subtypes, we have identified EP4 as the receptor that mediates bone formation in response to this agent. Consistently, bone formation was induced in wild-type mice by infusion of an EP4-selective agonist and not agonists specific for other EP subtypes. In culture of bone marrow cells from wild-type mice, PGE2 induced expression of core-binding factor alpha1 (Runx2/Cbfa1) and enhanced formation of mineralized nodules, both of which were absent in the culture of cells from EP4-deficient mice. Furthermore, administration of the EP4 agonist restored bone mass and strength normally lost in rats subjected to ovariectomy or immobilization. Histomorphometric analysis revealed that the EP4 agonist induced significant increases in the volume of cancellous bone, osteoid formation, and the number of osteoblasts in the affected bone of immobilized rats, indicating that activation of EP4 induces de novo bone formation. In addition, osteoclasts were found on the increased bone surface at a density comparable to that found in the bone of control animals. These results suggest that activation of EP4 induces bone remodeling in vivo and that EP4-selective drugs may be beneficial in humans with osteoporosis.

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Figures

Figure 1
Figure 1
Absence of PGE2-induced bone formation in EP4−/− mice. (a) PGE2-induced bone formation in wild-type mice. Typical radiographs (Left; arrowheads indicate the site of infusion) and histological preparations (Right; bars = 1 mm) of eight mice injected with vehicle or with PGE2 at a dose of 800 nmol/kg per day are shown. (b) The dose dependence of the effect of PGE2 on callus formation in wild-type mice. Data are values from six mice per dose. *, P < 0.01; **, P < 0.001 vs. vehicle-treated control mice. (c) The effect of PGE2 on bone formation in EP-deficient mice. Typical radiographs (Left) and hematoxylin/eosin staining (Right) of the treated femur from each mouse strain infused with PGE2 at a dose of 800 nmol/kg per day for 6 weeks are shown. Six mice per each strain were used in the analysis with reproducible results. (Bars = 1 mm.)
Figure 2
Figure 2
EP4 mediates mineralized nodule formation and Cbfa1 expression in cultured bone marrow cells. (a) EP4-mediated enhancement of mineralized nodule formation by PGE2. (Upper) von Kossa staining of mineralized nodules in bone marrow cell culture from wild-type C57BL/6 (WT) and EP4−/− mice in the presence of either vehicle or 100 nM PGE2. Nuclei were counterstained with neutral red in the EP4−/− cell cultures. Typical results of six independent cultures are shown. (Lower) The concentration dependence of the effect of PGE2 on mineralized nodule formation. Data are values from six experiments. *, P < 0.001 vs. vehicle-treated control. n.d., not detected. (b) Immunoblot analysis for Cbfa1 expression in bone marrow cells. Results of wild-type (WT) and EP4−/− cells cultured for indicated days in the absence or presence of 100 nM PGE2 are shown. (c) Northern blot analysis for Cbfa1 mRNA abundance. Wild-type bone marrow cells cultured in the absence or presence of 100 nM PGE2 for 7 days were subjected to Northern blot with probes specific to Cbfa1 or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAs. (d) Effect of PGE2 on the number of Cbfa1-positive cells. Bone marrow cells from wild-type mice cultured for 4 days in the absence or presence of 100 nM PGE2 were stained with anti-mouse Cbfa1 antibody and visualized by the ABC method. The number of positive cells per culture was determined (n = 10). *, P < 0.001 vs. vehicle-treated culture.
Figure 3
Figure 3
Selective induction of bone formation by an EP4 agonist. Radiograph and histology of the femora treated with either the EP4 agonist AE1–329 (a), EP1 agonist DI-004 (c), EP2 agonist AE1–259 (d), or EP3 agonist AE-248 (e) are shown. Typical findings with a dose of 800 nmol/kg per day are shown. (Bars = 1 mm.) In b, the dose dependence of the effects of AE1–329 is shown. Data are from four animals per each dose. *, P < 0.01; **, P < 0.001 vs. vehicle-treated control.
Figure 4
Figure 4
Prevention of bone loss and restoration of bone mass and strength by the EP4 agonist ONO-4819 in OVX rats. (a) Structure of AE1–329 (Left) and ONO-4819 (Right). (b) Bone architecture of OVX rats treated with ONO-4819. Seventy days after surgery, the right femur was isolated from a sham-operated rat as well as from OVX rats injected either with vehicle or two different doses of ONO-4819 and was subjected to analysis by x-ray-computed tomography. The three-dimensional structure was constructed by piling up images, and typical architecture of the metaphysis of each bone is presented. (c) Effects of ONO-4819 on bone density in OVX rats. OVX rats were injected s.c. with either vehicle or the indicated doses of ONO-4819 three times per day, either beginning on the day of surgery or day 20 after ovariectomy (post). Seventy days after the surgery, the left femur was isolated and subjected to analysis of bone density (n = 8 per group). #, P < 0.05 vs. the sham-operated group; *, P < 0.05 vs. control vehicle-treated OVX group (Dunnett test); +, P < 0.05 vs. control vehicle-treated OVX group (Student's t test). (d) Effects of ONO-4819 on bone strength in OVX rats. OVX rats were treated either by i.v. infusion of ONO-4819 at a rate of 30 or 100 ng/kg per min (≈15 and 50 nmol/kg per day) or by s.c. injection of the drug at a dose of 30 μg/kg three times per day (≈200 nmol/kg per day). Seventy days after surgery, rats were killed, and the fourth lumbar body was isolated and subjected to a compression test. No significant difference in the size of the bone was detected among the groups. Data are from eight rats per group. *, P < 0.05 vs. control vehicle-treated OVX group; *, P < 0.01 vs. control vehicle-treated OVX group. (e) Histology of the bone. Hematoxylin/eosin staining of the decalcified transverse sections of the epiphysis of the femur of sham-operated rats or OVX rats with ONO-4819 infusion is shown. (Bars = 100 μm.)
Figure 5
Figure 5
Effects of ONO-4819 on immobilization-induced bone loss. Rats immobilized in the left hind limb (Immob) were infused systematically for 2 h twice per day with vehicle or the indicated doses of ONO-4819. After 14 days, the left tibia was isolated and subjected to analysis of bone density (n = 9–14 per group). +, P < 0.01 vs. the sham-operated group; *, P < 0.01 vs. control vehicle-treated immobilized group.
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