Introduction

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Prostaglandins, especially PGE₂, have multiple effects on bone, including stimulation of both resorption and formation (Raisz at al., 1993; reviewed in Bergmann and Schoutens, 1995). PGE₂ administered to rats in vivo increases cortical as well as trabecular bone mass (Jee et al., 1985, 1987; Mori et al., 1990; Suponitzky and Weinreb, 1998). PGE₁, an alternate agonist with the same activity spectrum as PGE₂, was shown to stimulate bone formation and cause hyperostosis in infants (Ueda et al., 1980; Ringel et al., 1982).

Despite extensive documentation of in vivo bone anabolic effects, the cellular and molecular mechanisms that mediate PGE₂ action remain unclear. In organ culture of fetal rat calvaria, PGE₂ stimulates DNA synthesis in the periosteum, but suppresses collagen production (Raisz and Koolmans-Beynen, 1974). In the mouse MC3T3-E1 osteoblastic cell line, low concentrations of PGE₂ increase cell proliferation, and high concentrations stimulate differentiation (Hakeda et al., 1986). These effects correlate with an increase in intracellular calcium and cAMP, respectively. In cultures of adult rat calvaria cells, PGE₂ stimulates nodule formation, via a Ca²⁺ dependent pathway (Kaneki et al., 1999). In rat RP-11 periosteal cells (Machwate et al., 1998), PGE₂ increases cell number in vitro by suppressing apoptosis, without affecting proliferation. Similar effects were obtained using PGE₁ and forskolin, suggesting mediation via increased cAMP. It is thus unclear which of these biological responses and intracellular signaling pathways are more relevant to the bone anabolic effects of PGE₂ in vivo. Local administration of PGE₂ or E₁ into long bones in rats stimulates new bone formation (Jee et al., 1985), suggesting that PGE₂ acts directly on bone tissue to induce osteogenesis. PGEs bind to four subtypes of cell-surface receptors, EP_1-4 (reviewed in Narumiya et al., 1999; Sugimoto et al., 2000). These receptors belong to the G protein-coupled seven transmembrane domain family of receptors and activate either adenylate cyclase or phospholipase C (PLC). EP₄ and EP₂ activate adenylate cyclase, EP₁ activates PLC, and EP₃ inhibits adenylate cyclase, although EP₃ C-terminal splice variants can activate adenylate cyclase or PLC when expressed in recombinant systems. Prostaglandin receptors are expressed in a wide variety of cells and tissues (reviewed in Narumiya et al., 1999; Sugimoto et al., 2000). In MC3T3-E1 osteoblastic cells, PGE₂ stimulates both cAMP and phosphatidylinositol signal transduction pathways (Hakeda et al., 1986). Accordingly, both EP₁ and EP₄ were found to be expressed in these cells (Suda et al., 1996). In addition, EP₁, EP₂, and EP₄ were found to be expressed in preosteoblasts and osteoblasts in fetal bone tissues by in situ hybridization (Kasugai et al., 1995). EP₃ was found to be expressed in perichondrial cells. Analysis of the role of the individual EP subtypes in PGE₂ action on bone has been relatively limited because of the lack of specificity and limited efficacy of available agonists and antagonists for these receptors (Ono et al., 1998; Kozawa et al., 1998). However recent findings (Pan et al., 1998), using mice deficient in EP receptors, showed that those lacking EP₂ or EP₄ have defects in bone metabolism. Interestingly, the EP₄ deficient mice showed a marked decrease in histomorphometric parameters of bone formation as compared with the EP₂ deficient mice. In addition, we found, by Northern blot analysis, that only EP₄, but not EP₂, was detected in adult bone tissue. Together these data suggest that among the EP receptors, EP₄ may play a more predominant role in bone anabolic action of PGE₂.

In this study, we examined the effect of an EP₄ specific antagonist, EP₄A, on bone formation induced by PGE₂ in young rats. We found that EP₄A suppresses the increase in bone mass induced by PGE₂. This effect is accompanied by a reduction in the extent of calcein-labeled surface and trabecular number. Our data suggest that EP₄ is the main receptor through which PGE₂ induces bone formation in rats.

	Materials and Methods

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Prostanoid Receptor Radioligand Binding Assays. EP₄A [4'-[3-butyl-5-oxo-1-(2-trifluoromethyl-phenyl)-1,5-dihydro-[1,2,4]triazol-4-ylmethyl]-biphenyl-2-sulfonic acid (3-methyl-thiophene-2-carbonyl)-amide] was synthesized in Merck Research Laboratories. Prostanoid receptor radioligand binding assays were conducted as described previously for the human (Abramowitz et al., 2000) and rat receptors (Boie et al., 1997).

cAMP Measurements. RP-1 periosteal cells, like the RP-11 cells (Machwate et al., 1998), are spontaneously immortalized from primary cultures of periosteal cells from 4-week old Sprague-Dawley rat tibia and are cultured in DMEM (Life Technologies, Gaithersburg, MD) with 10% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS). These cells do not express osteoblast phenotypic markers in early culture, but upon confluence, they express several osteoblast markers: type I collagen, alkaline phosphatase, and osteocalcin.

Apoptosis. RP-1 cells were plated at 50,000 cells/cm² in 24-well plates (Costar, Cambridge, MA) and were cultured for 2 days in DMEM supplemented with 10% FBS. Cells were cultured for 24 h in DMEM supplemented with 2% FBS in the presence or absence of PGE₂ (0.1 µM), or in the presence of a combination of PGE₂ (0.1 µM) and EP₄A (10 µM). For analysis of apoptosis, the cells were trypsinized (0.25% trypsin, 1 mM EDTA) and single cell suspensions (1-2 million cells/well) were prepared. The cells were washed twice in Ca²⁺/Mg²⁺-free phosphate-buffered saline (PBS) and fixed in ethanol/PBS (3: 1 v/v) for 30 min. After centrifugation, cells were washed in PBS and processed for terminal deoxynucleotidyl transferase dUTP nick-end labeling staining according to the manufacturer's recommendations (Oncor, Gaithersburg, MD). Briefly, cells were incubated with nucleotide terminal transferase in the presence of dioxygenin-11-dUTP. Labeled cells were identified using an anti-digoxigenin, phycoerythrin-conjugated antibody. As control, the samples were exposed to the same mixture excluding the terminal transferase. Staining for annexin-V was analyzed with a FACScan flow cytometer (Becton Dickinson, San Francisco, CA). The red fluorescence was excited at 488 nm by the Argon laser beam. The data acquisition and analysis were performed using cellQuest software (Becton Dickinson, San Francisco, CA).

In Vivo Studies. A total of 40 male Sprague-Dawley rats (Taconic, Germantown, NY), 5-6 weeks old, weighing an average of 135 g at the start of the experiment, were randomly assigned to four groups (n = 10). One group was vehicle-treated (10% ethanol in sterile water), one group was treated with PGE₂ (6 mg/kg/day), one group with the EP₄ antagonist (EP₄A, 10 mg/kg/day), and the last group was treated with PGE₂ in combination with the EP₄ antagonist (EP₄A was given 45 min before PGE₂). All animals were treated for 12 days by daily intraperitoneal (i.p.) injection. Two days before sacrifice, all animals were given calcein (i.p., 10 mg/kg BW) to label the sites of active mineralization. At sacrifice, the animals were weighed then euthanized by CO₂ inhalation. Blood was collected by cardiac puncture, and tibiae were dissected and processed for histomorphometric analysis. The internal animal experimentation committee approved all protocols.

Plasma Biochemistry. Blood samples were obtained by cardiac puncture and plasma was immediately frozen. The plasma content of osteocalcin was determined by radioimmunoassay using a commercially available kit, according to the manufacturer's recommendations (Immunotopics International, San Clemente, CA).

Histomorphometric Analysis. Tibiae were dissected free from soft tissue, fixed in 10% phosphate-buffered formaldehyde, dehydrated in ethanol, and embedded undecalcified in methylmethacrylate (Baron et al., 1983). Longitudinal sections (5 µm) were cut with a Polycut S microtome (Reichert Jung, Heidelberg, Germany) and examined without further staining for dynamic histomorphometry, or stained with Masson's trichrome for static histological measurements. All histomorphometric measurements were carried out in cancellous bone with a semiautomatic image analysis system (System IV; Bioquant, Nashville, TN). Histomorphometric indices were measured in the proximal metaphyseal area (4 mm²) at a distance of 500 µm from the growth plate as described previously (Parfitt et al., 1983). Trabecular bone volume is expressed as the amount of bone within the spongy space. The mineralizing surface (MS/BS) is calculated as the sum of length of calcein labels and expressed in percent of the bone surface. Trabecular number is the number of bone trabeculae present in the proximal metaphysis within the area of measurement.

Statistical Analysis. Statistical analyses of the data were performed using the statistical package Statview (Abacus Concepts Inc., Berkeley, CA). Differences between treatment groups were tested by one-way ANOVA and unpaired two-tailed Student's t test. P values less than 0.05 at 95% confidence level were considered significant.

	Results

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EP₄A Binds Selectively to EP₄ and Antagonizes the Effects of PGE₂ on RP-1 Periosteal Cell Line. EP₄A (Fig. 1A) is a high-affinity EP₄ prostanoid receptor selective antagonist. It effectively competes with [³H]PGE₂ binding to both human and rat recombinant EP₄, with K_i values of 0.024 and 0.032 µM, respectively (Table 1 and Fig. 1B). EP₄A is selective for human EP₄ over all other members of the human prostanoid receptor family (EP₁, EP₂, EP₃, DP, FP, and IP). In addition, EP₄A is at least 200-fold more selective for rat EP₄ than the rat EP₁, EP₂, and EP₃ subtypes (Table 1). To determine whether EP₄A acts as an antagonist at rat EP₄, we used RP-1 periosteal cells, which express EP₄ protein (Weinreb et al., 2001) and in which PGE₂ increases cAMP intracellular levels. RP-1 periosteal cells were treated for 10 min with PGE₂ alone or in combination with increasing concentrations of EP₄A, then lysed and intracellular cAMP was measured by radioimmunoassay. As shown in Fig. 2A, PGE₂ (0.1 µM) increases intracellular cAMP more than 6-fold in this cell line. Treatment with EP₄A inhibits PGE₂-induced cAMP increases with an IC₅₀ value of 0.1 µM. To determine whether EP₄A interferes with PGE₂-independent accumulation of cAMP, we tested the effect of EP₄A on intracellular cAMP increased by forskolin. Figure 2B shows that EP₄A has no effect on intracellular cAMP induced by forskolin, suggesting that the antagonistic effect of EP₄A on PGE₂-mediated increase of intracellular cAMP is EP₄ mediated. These data are in agreement with previous results from Schild analysis demonstrating that EP₄A is a high-affinity competitive antagonist (K_B of 3-4 nM) opposing EP₄-induced increases in cAMP in HEK 293 cells expressing recombinant human EP₄. EP₄A did not antagonize cAMP increases induced by forskolin in EP₄-expressing or EP₄-deficient HEK 293 cells (data not shown). To further document the efficacy of EP₄A in antagonizing the PGE₂ effect, we tested the effect of EP₄A on apoptosis in these cells. Figure 2C shows that, as previously reported for the related RP-11 cells (Machwate et al., 1998), PGE₂ (0.1 µM) suppresses apoptosis measured by annexin-V binding. Cotreatment with EP₄A (10 µM) completely reverses the antiapoptotic effect of PGE₂. Together with the receptor binding data, these functional data suggest that EP₄A acts as a specific antagonist for prostanoid receptor EP₄.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Chemical structure of the EP4 antagonist EP4A and associated radioligand binding data. A, chemical structure of EP4A. B, competition curves for EP4A binding to HEK 293 cell membranes expressing recombinant human or rat EP4. Equilibrium competition binding assays were performed as described under Materials and Methods. Curves are representative of independent experiments with each data point a mean of duplicates.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   EP4A suppresses the effects of PGE2 on intracellular cAMP accumulation and apoptosis in RP-1 periosteal cells. RP-1 periosteal cells were treated with 0.1 µM PGE2 (A) or 1 µM forskolin (B) in the presence or absence of EP4A (0.01-10 µM). Intracellular cAMP was measured by radioimmunoassay after 10 min of treatment. C, RP-1 periosteal cells were cultured in medium containing low FBS (2%, v/v) in the presence or absence of 0.1 µM PGE2 alone or in combination with 10 µM EP4A. RP-1 periosteal cells were trypsinized and exposed to annexin-V. The percentage of cells bound to annexin-V was quantitated by fluorescence activated cell-sorting (FACS) to determine the percentage of apoptotic cells. Data are reported as mean ± S.D. and are from three experiments.

EP₄A Reverses PGE₂-Inceased Bone Formation in Rats. PGE₂ was administered at 6 mg/kg/day as described under Materials and Methods. PGE₂ has been known to induce diarrhea and decrease body weight. We therefore monitored the animals and evaluated if EP₄A interfered with this effect. We observed that diarrhea occurred in the groups treated with PGE₂ regardless of the presence of EP₄A. Furthermore, we found that treatment with EP₄A does not effect body weight loss induced by PGE₂, probably because of the diarrhea induced by PGE₂ acting on the intestine (Fig. 3). These data suggest that prostaglandin receptor EP₄ may not play a major role in this effect.

View larger version (25K): [in this window] [in a new window]   Fig. 3.   EP4A does not affect PGE2-induced decreased rat body weight. Five- to 6-week-old rats were randomized and treated with PGE2 (6 mg/kg/day) or EP4A (10 mg/kg/day) either alone or in combination. Animals were weighed after 12 days of treatment. Data are reported as mean ± S.E.M. *p < 0.01 versus vehicle and EP4A-treated groups

View larger version (27K): [in this window] [in a new window]   Fig. 4.   EP4A reverses PGE2- induced increased serum osteocalcin. Five- to 6-week-old rats were treated with PGE2 (6 mg/kg/day) or EP4A (10 mg/kg/day) or in combination for 12 days. Osteocalcin levels were determined in serum by radioimmunoassay. Data are reported as mean ± S.E.M. *p < 0.01 versus vehicle-treated group; **p < 0.05 versus PGE2-treated group

View larger version (36K): [in this window] [in a new window]   Fig. 5.   EP4A suppresses PGE2-induced increased trabecular bone volume, extent of calcein-labeled bone surface and trabecular number. Five- to 6-week-old rats were treated with PGE2 (6 mg/kg/day) or EP4A (10 mg/kg/day) or in combination for 12 days. The proximal tibial metaphysiswas embedded undecalcified. Frontal sections (5 µm) were stained with Masson's trichrome and subjected to quantitative histologic analysis. A, photomicrographs representative of the area of secondary spongiosa from which data were collected. The bone is stained blue, and the marrow stroma stained red. Scale bar, 0.1 mm. B, calcein-labeled surface, MS/BS (calcein labeled surface as a percentage of total bone surface). C, trabecular or cancellous bone volume, BV/TV (cancellous bone volume as a percentage of total bone volume). D, trabecular number, Tb. N, trabecular number per square millimeter of total tissue volume). Data are reported as mean ± S.E.M. *p < 0.01 versus vehicle-treated group; **p < 0.05 versus PGE2-treated group

	Discussion

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The present study demonstrates that pharmacological inactivation of prostanoid receptor EP₄ with EP₄A suppresses PGE₂-induced bone formation in vivo. PGE₂ and its analog PGE₁ are potent inducers of osteogenesis in humans (Ueda et al., 1980; Ringel et al., 1982) and animals (Jee et al., 1985, 1987; Mori et al., 1990; Suponitzky and Weinreb, 1998); however, the EP receptor that mediates osteogenic effects of PGE₂ has not been identified previously. In vitro studies did not provide conclusive evidence as to which EP subtype (EP₁, EP₂, EP₃, or EP₄) mediates the anabolic effects of PGE₂. This is mainly because PGE₂ has variable in vitro effects, depending on the osteoblastic cell type used (Raisz and Koolmans-Beynen, 1974; Hakeda et al., 1986; Kaneki et al., 1999), as stated in the introduction. In addition, the agonists and/or antagonists used so far to study PGE effects on bone were not sufficiently selective for the individual EP subtypes (Kozawa et al., 1998; Ono et al., 1998).

The genetic inactivation of EP subtypes in mice has provided evidence that prostanoid receptor EP₄ mediates PGE₂-induced bone resorption in mice (Ono et al., 1998; Miyaura et al., 2000; Suzawa et al., 2000). Indeed, studies of osteoclast formation in vitro showed that induction of this process depends on the presence of EP₄ in osteoblastic cells. These data are supported by recent in vivo findings showing that PGE₂-increased bone resorption is abrogated in these EP₄ deficient mice (Perry et al., 2000). On the other hand, another study, using EP₂ deficient mice, showed that EP₂ mediates, at least partially, the induction of bone resorption induced by thyroparathyroidectomy (Tomita et al., 1999). As mentioned above, PGE₂ can stimulate both bone resorption and formation in vivo. These effects are species specific, which should be considered when interpreting the data from gene deletion studies in mice. In mice, PGE₂ is a strong stimulator of bone resorption compared with rats and humans, in which PGE₂ predominantly increases bone formation. A pharmacological approach aimed at specifically targeting EP subtypes, therefore, is better suited for identifying which receptors mediate PGE₂ effects on bone in rats.

EP₄A, the EP₄ antagonist used in this study, is highly selective for EP₄. This compound displays a K_i value for binding to rat EP₄ that is at least 225-fold lower than the K_i values determined at rat EP₁, EP₂, and EP₃. We also showed pharmacologically that EP₄A acts as a PGE₂ antagonist, in that it dose-dependently inhibited PGE₂-induction of intracellular cAMP formation in a responsive cell line, RP-1. In addition, EP₄A reverses a cAMP-mediated biological effect of PGE₂ in these cells, the suppression of apoptosis. Pharmacokinetic studies (data not shown) showed that EP₄A reaches 1 µM in the blood 1 h after intraperitoneal injection with an estimated half-life of 3 h. EP₄A, therefore, is a highly selective antagonist for EP₄ with appropriate pharmacokinetic properties for in vivo studies evaluating pharmacologically the function of EP₄ in PGE₂ action on bone formation. Treatment with EP₄A suppressed PGE₂-induced increases in trabecular bone volume, suppressed serum osteocalcin and reduced the extent of calcein-labeled bone surface and trabecular number. These findings indicate that the reduction in bone volume is most likely a result of decreased bone formation.

The cellular mechanisms that mediate the bone anabolic effects of PGE₂ are still unclear and require further study. We have previously shown that PGE₂ increases periosteal cell number in vitro by suppressing apoptosis, without affecting proliferation. Similar effects were obtained using PGE₁ and forskolin, indicating cAMP mediation (Machwate et al., 1998). Interestingly, parathyroid hormone, which also stimulates bone formation and intracellular cAMP accumulation, was found to increase osteoblast number in vivo without increasing proliferation (Dobnig and Turner, 1995; Jilka et al., 1999). PGE₂, which may act via a mechanism similar to that of parathyroid hormone, may prolong the life span of bone forming cells and thereby increase their number. The role of apoptosis and cellular life span in the anabolic effect of PGE₂ in vivo remains to be documented. Future in vivo studies, potentially using EP₄A, will be necessary to determine the extent to which regulation of osteoblast apoptosis plays a role in PGE₂ bone anabolic effects.

This is the first study demonstrating the use of a selective PGE₂ receptor antagonist, targeting EP₄, to elucidate the role of EP₄ in in vivo effects of PGE₂ on bone. Further studies using a similar approach with an EP₄ agonist and ligands that target the other EP receptors are necessary to evaluate whether EP₄ is the only or major prostanoid receptor that mediates the bone anabolic effects of PGE₂ in rats.