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Endocrinology Vol. 141, No. 4 1554-1559
Copyright © 2000 by The Endocrine Society

ARTICLES

The Role of Prostaglandin E Receptor Subtypes (EP1, EP2, EP3, and EP4) in Bone Resorption: An Analysis Using Specific Agonists for the Respective EPs

Tetsuo Suzawa, Chisato Miyaura, Masaki Inada, Takayuki Maruyama, Yukihiko Sugimoto, Fumitaka Ushikubi, Atsushi Ichikawa, Shuh Narumiya and Tatsuo Suda

Department of Biochemistry, Showa University School of Dentistry (Te.S., C.M., M.I., Ta.S.), 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555; Department of Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life Science (C.M., M.I.), Tokyo 192-0392; Discovery Research Laboratory I, Minase Research Institute, Ono Pharmaceutical Co. Ltd. (T.M.), Osaka 618-8585; and Department of Physiological Chemistry, Faculty of Pharmaceutical Science (Y.S., A.I.), Kyoto 606-8501; and Department of Pharmacology, Faculty of Medicine (F.U., S.N.), Kyoto University, Kyoto 606-8501, Japan

Address all correspondence and requests for reprints to: Dr. Tatsuo Suda, Department of Biochemistry, Showa University School of Dentistry, 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
PGE2 functions as a potent stimulator of bone resorption. The action of PGE2 is thought to be mediated by some PGE receptor subtypes present in osteoblastic cells. In this study, we examined the involvement of PGE receptor subtypes, EP1, EP2, EP3, and EP4, in PGE2-induced bone resorption using specific agonists for the respective EPs. In mouse calvaria cultures, EP4 agonist markedly stimulated bone resorption, but its maximal stimulation was less than that induced by PGE2. EP2 agonist also stimulated bone resorption, but only slightly. EP1 and EP3 agonists did not stimulate it at all. RT-PCR showed that osteoblastic cells isolated from newborn mouse calvaria expressed all of the EPs messenger RNA (mRNA). Both EP2 agonist and EP4 agonist induced cAMP production and the expression of osteoclast differentiation factor (ODF) mRNA in osteoblastic cells. Simultaneous addition of EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression. In mouse bone marrow cultures, EP2 and EP4 agonists moderately induced osteoclast formation, but the simultaneous addition of the two agonists cooperatively induced it, similar to that by PGE2. In calvaria culture from EP4 knockout mice, a marked reduction in bone resorption to PGE2 was found. In EP4 knockout mice, EP4 agonist failed to induce bone resorption, but EP2 agonist slightly, but significantly, induced bone resorption. These findings suggest that PGE2 stimulates bone resorption by a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
PGs ARE produced in bone mainly by osteoblasts and stimulate bone resorption in vitro (1, 2, 3). Among several PGs produced, PGE2 is a major product, and its production by osteoblasts is regulated by several cytokines, including interleukin-1 (IL-1). We previously reported that PGE2 stimulated adenylate cyclase in osteoblasts to accumulate cellular cAMP, induced osteoclast formation in mouse bone marrow cultures, and stimulated bone resorption in calvaria cultures (4, 5, 6). Addition of an inhibitor of A kinase, H89, suppressed bone resorption induced by PGE2, and (Bu)2cAMP greatly stimulated bone resorption. Therefore, it is suggested that PGE2 stimulates bone resorption via a cAMP-dependent mechanism. However, the PGE receptor(s) mediating PGE-induced bone resorption remains to be elucidated.

The action of PGE2 is mediated by rhodopsin-type receptors specific to PGs. There are four subtypes of PGE receptors, designated EP1, EP2, EP3, and EP4, that are encoded by different genes and expressed differently in each tissue (7, 8, 9, 10). In addition, mouse EP3 was reported to have three isoforms (EP3{alpha}, EP3ß, and EP3{gamma}) with different carboxyl-terminal tails produced by alternative splicing (11, 12). The intracellular signaling differs among the receptor subtypes; EP1 is coupled to Ca2+ mobilization, and EP3 inhibits adenylate cyclase, whereas both EP2 and EP4 stimulate adenylate cyclase (13, 14, 15). To identify the physiological function of each EP receptor subtype, we generated mice lacking respective receptors by homologous recombination (16, 17, 18). Loss of EP4 was not lethal in utero, but most EP4-/- neonates died within 72 h after birth due to patent ductus arteriosus, suggesting that EP4 played a role in the regulation of the patency of this vessel (16, 19). On the other hand, EP3-/- mice failed to show a febrile response to various pyrogens, suggesting that PGE2 mediates fever generation by acting on EP3 (17).

Recently, we used EP1, EP2, EP3, and EP4 knockout mice to analyze the bone-resorbing activity of PGE2 in calvaria cultures (20). A significant reduction in bone resorption in response to PGE2 was detected only in calvaria cultures from EP4 knockout mice, suggesting the importance of EP4 in PGE2-induced bone resorption (20). However, some of the bone-resorbing activity induced by PGE2 remained in EP4 knockout mice (20). Thus, other EPs (EP1, EP2, or EP3) could also be involved in PGE2-induced bone resorption.

More recently, we cloned osteoclast differentiation factor (ODF), which was expressed on the surface of osteoblasts and bone marrow stromal cells treated with bone-resorbing factors such as IL-1, 1{alpha},25-dihydroxyvitamin D3 [1{alpha},25(OH)2D3], PTH, and PGE2 (21). ODF was identical to receptor activator of nuclear factor-{kappa}B ligand, tumor necrosis factor-related activation-induced cytokine, and osteoprotegerin ligand, which were cloned independently in other studies (22, 23, 24). ODF is essential for the differentiation of osteoclast progenitors into mature osteoclasts (21). Thus, monitoring ODF induction in osteoblasts appears to be a suitable measure for PGE-mediated bone resorption.

In the present study, we examined which EP(s) mediated the bone-resorbing activity of PGE2, using specific agonists for the respective EPs. The actions of the EP agonists in bone resorption were also analyzed using EP4 knockout mice. We report here that the bone-resorbing activity of PGE2 is mediated mainly by EP4 and partially by EP2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and drugs
Newborn (C57BL/6 strain) mice were obtained from SLC, Inc. (Shizuoka, Japan). Mice lacking EP4 were generated, and homozygote and wild-type mice of the F2 progeny were used (16). To examine the genotype of each mouse, PCR analysis was performed on DNA extracted from the tail or brain of neonates, using the oligonucleotide primers designed to detect the EP4 locus and Neo cassette, as reported previously (16). PGE2 was obtained from Sigma (St. Louis, MO). EP1, EP2, EP3, and EP4 agonists were generated, and the specificity of the respective EP agonists was analyzed by measuring the binding affinity of the agonists to the respective EPs expressed in CHO cells (25, 26) (Table 1Go). PGE2 bound to all EPs. However, the EP agonists selectively bound to the respective EPs (Table 1Go). All other chemicals were of analytical grade.


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  		Table 1. Binding affinities of EP1, EP2, EP3, and EP4 agonists to the respective EPs expressed in CHO cells

 
Mouse calvaria cultures
One-day-old mice were killed, and their calvaria were aseptically isolated and dissected free of suture tissues. The calvaria were divided into halves and cultured for 24 h at 37 C under 5% CO2 in air in 0.12 ml BGJb medium (Life Technologies, Inc., Rockville, MD) containing 1 mg/ml BSA (fraction V, Sigma). After preculture for 24 h, each half-calvaria was transferred to fresh medium with or without the indicated reagents and cultured for an additional 72 h. Bone-resorbing activity was determined by measuring the concentration of calcium in the conditioned medium using a calcium kit (Calcium C-test Wako, Wako Pure Chemicals, Osaka, Japan). The activity was expressed as an increase in medium calcium. The activity was changed in parallel with the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts in cultured calvaria (4, 27).

Culture of primary mouse osteoblastic cells
Primary osteoblastic cells were isolated from 1-day-old mouse calvariae after five routine sequential digestions with 0.1% collagenase (Wako) and 0.2% dispase (Godo Shusei, Tokyo, Japan), as previously described (28). Osteoblastic cells collected from fractions 3–5 were combined and cultured in {alpha}MEM supplemented with 10% FCS at 37 C under 5% CO2 in air. Osteoblastic cells were cultured for 24 h in {alpha}MEM containing 1% FCS, then treated with PGE2 or the respective EP agonists.

Assay of cAMP production
To measure the amount of cAMP produced, osteoblastic cells were preincubated for 5 min at 37 C in {alpha}MEM containing 1 mM 3-isobuthyl-1-methylxanthine, then incubated for 7 min at 37 C with 10 µM of the respective EP agonists or PGE2. Cells were dissolved, and the content of cellular cAMP was determined using a cAMP enzyme immunoassay system (Amersham Pharmacia Biotech, Aylesbury, UK).

Mouse bone marrow cultures
Bone marrow cells were isolated from 6-week-old C57BL/6 mice and cultured in 0.5 ml {alpha}MEM containing 10% FCS at 1 x 106 cells/well in 24-well plates. Cultures were fed every 3 days by replacing 0.4 ml old medium with fresh medium. All reagents were added at the beginning of the culture, and each time the medium was changed. After being cultured for 7 days, cells adherent to the well surface were stained for TRAP and alkaline phosphatase. The number of TRAP-positive multinucleated cells containing three or more nuclei per cell was counted as osteoclasts.

Northern blot analysis
Total RNA was extracted from cultured mouse osteoblastic cells using the acid guanidium-phenol-chloroform method (28). For Northern blotting, 20 µg total RNA were resolved using electrophoresis on a 1% agarose-formaldehyde gel and transferred onto a nylon membrane, which was then hybridized with a 32P-labeled complementary DNA (cDNA) probe, as previously reported (28). A 946-bp fragment of mouse ODF cDNA and a 983-bp fragment of mouse glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA were prepared using RT-PCR and used as the respective probes (21, 29). The signals were densitometrically quantified using a Bioimage analyzer (BAS-2000, Fuji Photo Film Co., Ltd., Tokyo, Japan).

RT-PCR analysis
cDNA was synthesized from 10 µg total RNA by reverse transcriptase (Superscript II Preamplification System, Life Technologies, Inc., Grand Island, NY) and amplified using PCR. Primers used in PCR for EP1, EP2, EP3{alpha}, EP3ß, EP3{gamma}, and EP4 genes were: EP1, 5'-TTAACCTGAGCCTAGCGGATG-3' (sense primer; nucleotides 13–37) and 5'-CGCTGAGCGTATTGCACACTA-3' (antisense primer; nucleotides 662–682); EP2, 5'-CCACGATGCTCTCCTGCTGCTTAT-3' (sense primer; nucleotides 750–771) and 5'-CAGCCCCTTACACTTCTCCAATGA-3' (antisense primer; nucleotides 1257–1280); EP3{alpha}, 5'-TGACCTTTGCCTGCAACCTG-3' (sense primer, nucleotides 659–678) and 5'-AGCTGGAAGCATAGTTGGTG-3' (antisense primer; nucleotides 1017–1036); EP3ß, 5'-TGACCTTTGCCTGCAACCTG-3' (sense primer; nucleotides 659–678) and 5'-GACCCAGGGAAACAGGTACT-3' (antisense primer; nucleotides 1038–1057); EP3{gamma}, 5'-TGACCTTTGCCTGCAACCTG-3' (sense primer; nucleotides 659–678) and 5'-AGACAATGAGATGGCCTGCC-3' (antisense primer; nucleotides 1049–1068); and EP4, 5'-GGTCATCTTACTCATCGCCACCTCTC-3' (sense primer; nucleotides 1027–1052), 5'-TCCCACTAACCTCATCCACCAACAG-3' (antisense primer; nucleotides 1538–1562). The reaction condition for all PCRs was 30 cycles, denaturation at 94 C for 30 sec, annealing at 65 C for 30 sec, and extension at 75 C for 60 sec. PCR products were run on a 1% agarose gel and stained with ethidium bromide.

Statistical analysis
Statistical analysis was carried out using Student’s t test, and the data were expressed as the mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Bone-resorbing activities of EP1, EP2, EP3, and EP4 agonists
PGE2 markedly stimulated osteoclastic bone resorption in vitro. We previously reported that PGE2 acted on osteoblasts to elicit cAMP production and promoted osteoclast formation via a mechanism involving cAMP in mouse bone marrow cultures (1, 4). However, the PGE receptor subtype(s) mediating bone resorption remained unknown. To identify the responsible receptor subtype(s), specific agonists for the respective EPs were synthesized to examine their effects on bone resorption in vitro. The specificity of the agonists to the respective EPs was firstly defined from their binding affinity to each EP expressed in CHO cells (Table 1Go). EP1, EP2, EP3, and EP4 agonists selectively bound to EP1, EP2, EP3, and EP4, respectively, whereas PGE2 bound to all EPs. In mouse calvaria cultures, the EP4 agonist markedly stimulated bone resorption at 0.1–10 µM, but the maximal level was less than that of PGE2 (Fig. 1Go). The EP2 agonist (1–10 µM) also stimulated bone resorption, but only slightly. Neither EP1 nor EP3 agonist stimulated bone resorption. Simultaneous addition of the EP2 and EP4 agonists cooperatively induced bone resorption, suggesting that not only EP4, but also EP2, were involved in bone resorption induced by PGE2.



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  		Figure 1. Effects of EP1, EP2, EP3, and EP4 agonists on bone resorption in mouse calvaria cultures. Calvaria collected from 1-day-old C57BL/6 mice were cultured for 72 h with various concentrations of PGE2 ({diamond}), EP1 agonist ({square}), EP2 agonist ({triangleup}), EP3 agonist (•), EP4 agonist ({blacktriangleup}), and EP2 agonist plus EP4 agonist ({circ}). Conditioned media were collected, and the calcium content was measured. Bone-resorbing activity was expressed as the increase in medium calcium. Data were expressed as the mean ± SEM of 10 independent experiments.

 
Expression of EP1, EP2, EP3, and EP4 messenger RNAs (mRNAs) in osteoblasts
Expression of EPs mRNA was studied in primary osteoblastic cells isolated from newborn mouse calvaria using RT-PCR analysis with specific primers for the respective EPs. The messages for EP1 and EP4 were clearly detected in osteoblastic cells (Fig. 2Go). Expressions of EP2 mRNA and three isoforms of EP3 (EP3{alpha}, EP3ß, and EP3{gamma}) mRNA were also detected, but only slightly (Fig. 2Go).



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  		Figure 2. Expression of EP1, EP2, EP3, and EP4 mRNAs in mouse primary osteoblastic cells measured using RT-PCR. Total RNA was extracted from primary osteoblastic cells, and subjected to RT-PCR for EP1 (lane 1), EP2 (lane 2), EP3{alpha} (lane 3), EP3ß (lane 4), EP3{gamma} (lane 5), and EP4 (lane 6) mRNAs using the respective primers as described in Materials and Methods. M, Size marker.

 
Effects of EP agonists on cAMP production by osteoblasts
PGE2 acts on osteoblasts to elicit cAMP production, and (Bu)2cAMP stimulates bone resorption (1, 4). Therefore, it is critical to define the effects of the EP agonists on cAMP production by osteoblasts to analyze the EP(s) involving PGE2-induced bone resorption. As reported previously, PGE2 markedly stimulated cAMP production when osteoblastic cells were incubated for 7 min (Fig. 3Go). Both the EP2 agonist and the EP4 agonist enhanced cellular cAMP, but the levels were less than that of PGE2 (Fig. 3Go). Simultaneous addition of the EP2 and EP4 agonists additively stimulated cAMP production. Neither the EP1 agonist nor the EP3 agonist stimulated cAMP production by osteoblasts (Fig. 3Go). This suggests that cAMP production elicited by PGE2 is mediated by both EP2 and EP4.



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  		Figure 3. Effects of EP1, EP2, EP3, and EP4 agonists on cAMP production by mouse primary osteoblastic cells. Osteoblastic cells (5 x 104 cells/well) cultured in 24-well plates were incubated for 7 min with the respective agonists or with the EP2 agonist plus the EP4 agonist, then cellular cAMP was measured as described in Materials and Methods. Data were expressed as the mean ± SEM of four independent experiments.

 
Expression of ODF mRNA by EP2 and EP4 agonists in osteoblastic cells
As ODF is expressed on the surface of osteoblasts by bone-resorbing factors, including PGE2, the effects of four EP agonists on the expression of ODF mRNA in osteoblastic cells was examined. At 3 h, PGE2 greatly induced ODF mRNA expression (Fig. 4Go). Both the EP2 agonist and the EP4 agonist induced ODF mRNA expression, and the simultaneous addition of the two agonists cooperatively enhanced it (Fig. 4Go). Neither the EP1 agonist nor the EP3 agonist stimulated ODF mRNA expression.



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  		Figure 4. Effects of EP1, EP2, EP3, and EP4 agonists on the expression of ODF mRNA in mouse primary osteoblastic cells. A, Mouse primary osteoblastic cells (1.0 x 106 cells) were cultured with or without the respective agonists for 3 h, and total RNA was extracted. Northern blotting was performed using 32P-labeled cDNA probes for ODF and glyceraldehyde-3-phosphate dehydrogenase (G3PDH). B, Signals of ODF mRNA in A were subjected to a radioactive image analyzer (BAS 2000) and normalized with G3PDH mRNA levels to calculate the relative intensity. Lane 1, Vehicle (control); lane 2, 10 µM PGE2; lane 3, 10 µM EP1 agonist; lane 4, 10 µM EP2 agonist; lane 5, 10 µM EP3 agonist; lane 6, 10 µM EP4 agonist; lane 7, 10 µM EP2 agonist plus 10 µM EP4 agonist.

 
Cooperative effects of EP2 agonist and EP4 agonist on osteoclast formation
To define PGE receptor subtypes mediating osteoclastogenesis, the effects of EP agonists on osteoclast formation were examined in mouse bone marrow cultures. As reported previously, adding 10 µM PGE2 markedly induced TRAP-positive osteoclast-like cells (Fig. 5Go). The EP4 agonist moderately induced osteoclast formation. The EP2 agonist also elicited it, but only slightly. Simultaneous addition of both the EP2 agonist and the EP4 agonist greatly induced osteoclast formation, and the potency was similar to that of PGE2. Neither the EP1 agonist nor the EP3 agonist stimulated osteoclast formation (Fig. 5Go).



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  		Figure 5. Effects of EP1, EP2, EP3, and EP4 agonists on osteoclast formation in mouse bone marrow cultures. Mouse bone marrow cells were cultured with vehicle (control), 10 µM PGE2, 10 µM EP1 agonist, 10 µM EP2 agonist, 10 µM EP3 agonist, 10 µM EP4 agonist, or 10 µM EP2 agonist plus 10 µM EP4 agonist. After culture for 7 days, cells were stained with TRAP and alkaline phosphatase (A), and TRAP-positive multinucleated cells containing three or more nuclei per cell were counted (B).

 
Bone-resorbing activity of EP2 agonist and EP4 agonist in EP4 knockout mice
Finally, the effects of the EP2 agonist, the EP4 agonist, and PGE2 were compared on bone resorption in calvaria cultures collected from EP4-/- mice and wild-type mice. The EP4 agonist showed no bone resorption in EP4-/- mice, but the EP2 agonist slightly, but significantly, stimulated bone resorption (Fig. 6Go). Therefore, it is concluded that the PGE2-induced bone-resorbing activity detected in EP4-/- mice is expressed via EP2, indicating that PGE2 stimulates bone resorption mainly by EP4 and partially by EP2, and that the two EPs act in concert to elicit the full function of PGE2 in bone.



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  		Figure 6. Effects of the EP2 agonist and the EP4 agonist on bone-resorbing activity in calvaria collected from EP4-/- mice. Calvaria collected from EP4-/- mice and wild-type mice were cultured for 72 h with 10 µM PGE2, 10 µM EP2 agonist, or 10 µM EP4 agonist. Conditioned media were collected, and medium calcium was measured to monitor the bone-resorbing activity. Data were expressed as the mean ± SEM of eight independent experiments. *, P < 0.001, significantly different from the cultures treated with PGE2 (10 µM) in wild-type mice.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
To define which EP(s) was involved in PGE2-induced bone resorption, newly synthesized EP(s) agonists, specific to the respective EPs, were used. The EP4 agonist markedly stimulated bone resorption in mouse calvaria cultures. The EP2 agonist also stimulated bone resorption, but only slightly. Simultaneously adding the EP2 and EP4 agonists cooperatively induced bone resorption. Similarly, the EP2 and EP4 agonists cooperatively induced cAMP production and ODF mRNA expression in osteoblasts as well as osteoclast formation in bone marrow cultures. These findings suggest that PGE2 stimulates bone resorption via a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2.

Previous studies have shown that the distribution of EP1-EP4 is closely related to the tissue-specific biological functions of PGE in target organs (7, 8, 9, 10). All EPs were detected in osteoblastic cells isolated from mouse calvaria, and the expression level was relatively high in EP1 and EP4 (Fig. 2Go). It has been reported that EP1 and EP4 mRNAs are detected in mouse osteoblastic MC3T3-E1 cells (10). This is consistent with the finding that the EP4 agonist stimulates bone resorption in calvaria cultures. However, neither the EP1 agonist nor the EP3 agonist stimulated bone resorption. The intracellular signaling differs among EP1-EP4; EP1 is coupled to Ca2+ mobilization, EP3, especially EP3{alpha} and EP3ß, inhibits adenylate cyclase (13, 15), and EP2 and EP4 stimulate adenylate cyclase (14). EP3 agonist did not inhibit bone-resorbing activity induced by EP4 agonist or EP2 agonist (data not shown). Because EP3 has three isoforms, EP3{alpha}, EP3ß, and EP3{gamma}, further studies are needed to define the role of the respective EP3 isoforms in bone metabolism. The role of EP1 in bone metabolism is not clear either. Previous studies have shown that PGE2 stimulates bone formation in vivo (30, 31). Possible mechanisms of EPs in the anabolic effects of PGE in bone have to be examined in future studies.

In this study, EP2 and EP4 agonists similarly induced an increase in cAMP and ODF expression in osteoblasts. However, the bone-resorbing activity induced by EP4 agonist was more potent than that by EP2 agonist. Recently, Mano et al. (32) reported that butaprost, an EP2 agonist, inhibited the bone-resorbing activity of rabbit mature osteoclasts. These results suggest that EP2 signals, but not EP4 signals, suppress the bone-resorbing activity of mature osteoclasts, and that the difference in bone-resorbing activity between EP2 agonist and EP4 agonist was due to this specific effect of EP2 agonist in osteoclasts. Although further studies are needed to explain this issue, we suggest that the activation of A kinase by EP2 and EP4 in osteoblasts mediates the signals for bone resorption.

During the past decade we have studied the mechanism of differentiation of hemopoietic osteoclast precursors into osteoclasts. Osteoclast formation requires cell to cell contact between osteoblasts and hemopoietic osteoclast precursors (1). In 1998, we succeeded in the molecular cloning of ODF, expressed on the surface of osteoblasts treated with several bone-resorbing factors (21). Treatment with bone-resorbing factors such as 1{alpha},25(OH)2D3, PTH, IL-11, or PGE2 up-regulated the expression of ODF mRNA. ODF was identical to receptor activator of nuclear factor-{kappa}B ligand, tumor necrosis factor-related activation-induced cytokine, and osteoprotegerin ligand. In the present study, it was shown that the EP2 agonist and the EP4 agonist cooperatively induced ODF mRNA expression in osteoblasts. Furthermore, simultaneously adding the EP2 and EP4 agonists cooperatively induced osteoclast formation, and the potency was similar to that of PGE2. Kitazawa et al. (33) found a vitamin D receptor-responsive element and a glucocorticoid receptor-responsive element in the promoter region of the mouse ODF gene. Treatment with 1{alpha},25(OH)2D3 and dexamethasone increased luciferase activity in the ODF gene promoter (33). (Bu)2cAMP did not affect the promoter activity, suggesting that the signals via A kinase indirectly induced transcription of the ODF gene (33). Further studies are needed to define the mechanism of ODF expression induced by the EP2 and EP4 agonists and PGE2.

As reported previously, all EP1-/-, EP2-/-, EP3-/-, and EP4-/- mice are born at the predicted Mendelian frequency (16, 17, 18, 19). EP1-/-, EP2-/-, and EP3-/- mice grew normally, but most EP4-/- neonates died within 72 h after birth by patent ductus arteriosus (16, 19). In calvaria cultures from EP4-/- mice, a marked reduction in bone resorption in response to PGE2 was detected, but some of the bone-resorbing activity of PGE2 remained (20). The expression level of EP2 mRNA in osteoblasts collected from EP4-/- mice was similar to that from the wild-type mice (data not shown). Therefore, it is not likely that osteoblasts express a higher level of EP2 to compensate for the lack of EP4 in EP4-/- mice. The EP2 agonist moderately stimulated bone resorption not only in the wild-type mice but also in the EP4-/- mice, suggesting that the remaining bone-resorbing activity in EP4-/- mice was elicited by EP2. Therefore, it is concluded that the bone-resorbing activity induced by PGE2 is mainly via EP4 and partially via EP2.

PGE2 is a critical factor in bone resorption in several metabolic bone diseases, including osteoporosis, periodontal bone diseases, and rheumatoid arthritis (34, 35). PGE2 produced by osteoblasts is involved in the mechanism of osteoclast formation induced by cytokines such as IL-1 and IL-6. Indeed, indomethacin and NS-398, inhibitors of PG synthesis, strikingly suppressed osteoclast formation induced by IL-1 and IL-6 (5, 6). Therefore, it is likely that the specific antagonists for EP4 and/or EP2 may be useful to inhibit PGE-induced bone resorption. A trial developing specific EP antagonists is now being explored in our laboratories.

In conclusion, PGE2 stimulates bone resorption via a mechanism involving cAMP and ODF, which is mediated mainly by EP4 and partially by EP2. There are three regulatory phases for PGE2-induced bone resorption: the binding of PGE2 to EP4 and EP2, the increase in cAMP, and the ODF induction. Suppression of any of the three phases may provide a clue to bone resorption mediated by PGE.

Received September 29, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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