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Am J Physiol Endocrinol Metab 276: E376-E383, 1999;
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Vol. 276, Issue 2, E376-E383, February 1999

The anabolic effect of PGE2 in rat bone marrow cultures is mediated via the EP4 receptor subtype

M. Weinreb, A. Grosskopf, and N. Shir

Department of Oral Biology, Goldschleger School of Dental Medicine, Tel-Aviv University, Tel-Aviv 69978, Israel


    ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

Prostaglandin E2 (PGE2) is an anabolic agent in vivo that stimulates bone formation by recruiting osteoblasts from bone marrow precursors. To understand which of the known PGE2 receptors (EP1-4) is involved in this process, we tested the effect of PGE2 and various EP agonists and/or antagonists on osteoblastic differentiation in cultures of bone marrow cells by counting bone nodules and measuring alkaline phosphatase activity. PGE2 increased both parameters, peaking at 100 nM, an effect that was mimicked by forskolin and was abolished by 2',3'-dideoxyadenosine (an adenylate cyclase inhibitor) and was thus cAMP dependent, pointing to the involvement of EP2 or EP4. Consistently, 17-phenyl-omega -trinor PGE2 (EP1 agonist) and sulprostone (EP3/EP1 agonist) lacked any anabolic activity. Furthermore, butaprost (EP2 agonist) was inactive, 11-deoxy-PGE1 (EP4/EP2 agonist) was as effective as PGE2, and the PGE2 effect was abolished dose dependently by the selective EP4 antagonist AH-23848B, suggesting the involvement of EP4. We also found that PGE2 increased nodule formation and AP activity when added for the initial attachment period of 24 h only. Thus this study shows that PGE2 stimulates osteoblastic differentiation in bone marrow cultures, probably by activating the EP4 receptor, and that this effect may involve recruitment of noncommitted (nonadherent) osteogenic precursors, in agreement with its suggested mode of operation in vivo.

prostanoid receptors; osteoblast recruitment; bone nodules


    INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

PROSTAGLANDINS (PG) are multifaceted modulators of bone metabolism (42), and recent experimental animal studies have shown that PGE2, in particular, is a powerful systemic (3, 19, 20-23, 32, 33, 52) and local (31, 59) anabolic agent. The in vivo effects have been observed both in young (22, 23, 52) and older (3, 19, 20, 21, 32, 33) rats and dogs and also in humans (24, 51). The increased bone mass (cortical as well as cancellous) in PGE2-treated animals results primarily from a substantial production of new bone with apparent increased osteoblast number (23) and tetracycline-labeled bone surfaces (19, 22, 32). Despite vast, convincing evidence that PGE1 and PGE2 are potent anabolic agents in vivo, their mechanisms of action have not been established.

To gain insight into the modes of action of PGE2, we characterized a rat model in which 3- to 4-wk-old rats are injected daily with 6 mg/kg PGE2 for 2-3 wk. This treatment results in increased cancellous and cortical bone mass and increased mechanical strength of the femoral neck (49). This model enabled us to explore the mechanisms of this action. Strong evidence now suggests that PGE2 stimulates bone formation by recruiting new osteoblasts from their precursors. In young rats, most of the new bone produced is cancellous and therefore originates in bone marrow, and we believe, for the following reasons, that PGE2 induces bone marrow osteogenic precursors in these animals to differentiate into osteoblasts. 1) We recently found, using Northern analysis, that a single anabolic dose of PGE2 induces the expression of early-response genes, such as c-fos, c-jun, junB, and egr-1, in the tibia and calvaria of young rats as early as 15 min postinjection (54). Using in situ hybridization, we showed that the induced expression of these genes occurred in bone marrow cells. These data indicate that PGE2 activates multiple transcription factors within the bone marrow compartment, probably to stimulate the proliferation and/or differentiation of osteogenic precursors. 2) Testing this hypothesis directly, we showed that the osteogenic capacity of bone marrow (i.e., the size of the osteoprogenitor pool) is greatly enhanced by systemic treatment with PGE2 in vivo for 2 wk (55). For this purpose, we used an ex vivo bone marrow culture system that enables osteoblastic differentiation of osteogenic precursors belonging to the fibroblastic colony-forming unit population (17, 27, 30, 40). We showed that bone marrow from PGE2-treated rats yielded many more osteogenic colonies (nodules) and a greater alkaline phosphatase (AP) activity compared with bone marrow from vehicle-injected rats. Because each of these colonies is believed to originate from a single precursor cell, this means that PGE2 in vivo stimulates the osteogenic commitment of bone marrow precursors, as we had hypothesized.

We now seek to study the mechanisms by which PGE2 recruits osteoblasts from their marrow precursors and primarily which of the PGE2 receptors known today mediates the action of PGE2. Prostaglandins exert their actions on various cells in the body via specific cell surface receptors that are termed EP and have been divided into four subtypes (EP1-4) according to their relative sensitivity to a range of selective agonists and antagonists (7, 12, 36, 37). In recent years, the human, rat, and mouse EP1-4 receptors have been cloned and characterized (1, 2, 5, 16, 26, 38, 53). They all have seven transmembrane domains and are coupled to different G proteins and activate different secondary messenger systems, such as adenylyl cyclase or phospholipase C. Recent data point to the possibility that EP2 and EP4, the two receptors using the cAMP signal transduction system, are the most important receptors in the effects of PGE2 on bone cells. 1) Initial localization experiments in embryonic and neonatal mice showed that EP4 is the major form found in bone tissue, especially in preosteoblasts, with some EP3 expressed in perichondrium (18). In support of the role of EP4 in bone, we recently found by Northern analysis and in situ hybridization that it is also expressed in bone marrow cells of young adult rats (M. Weinreb, M. Machwate, N. Shir, M. Abramovitz, G. A. Rodan, and S. Harada, unpublished observations). Also, expression of EP4 (originally labeled erroneously as EP2; see Refs. 39 and 53) was found in neonatal rat calvaria by in situ hybridization and in rat bone marrow cultures with PCR (25). 2) In the majority of systems examined, the stimulatory effects of PGE2 are cAMP mediated (8, 10, 41, 46, 47, 58), pointing to the possible involvement of EP2 or EP4.

Therefore, the purpose of this study was to test which of the known EP receptors mediates the anabolic action of PGE2 in vitro by using an osteogenic bone marrow culture system in which PGE2 is anabolic and by using an array of EP agonists and antagonists.


    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

All animal protocols were approved by the animal experimentation committee. Sprague-Dawley rats, 6 or 7 wk old, were killed by CO2, their femora were excised and defleshed, and the epiphyses were removed. Bone marrow was flushed out, and a single cell suspension was achieved by repeated pipetting. Cells from each femur were cultured in 6-well plates (Nunc) at a density of ~2 × 107 cells/well in a medium containing alpha -MEM + 13% fetal calf serum (all reagents except where noted were from Biological Industries, Beit-Haemek, Israel) + 2 mM glutamine + 100 U/ml penicillin + 100 µg/ml streptomycin + 12.5 U/ml nystatin + 10 mM beta -glycerophosphate + 50 µg/ml ascorbic acid (Merck, Darmstadt, Germany) + 10 nM dexamethasone (Dex, Sigma). Wells were arranged in triplicates, and the different compounds were added to the culture medium. After an attachment period of 24 h, nonadherent cells were removed by a PBS (phosphate-buffered saline) rinse, and cultures were maintained in 7% CO2 at 37°C for 21 days, with medium changes twice weekly. At the end of the culture period, cultures were rinsed in PBS, fixed in a 1:1:1.5 solution of 10% Formalin-methanol-water for 2 h, and stained with the Von Kossa method for mineralized nodules (27). Mineralized nodules (completely or partially stained black) and nonmineralized nodules (stained yellow) were counted under a magnifying glass over transmitted light, and the relative proportion of mineralized nodules of the total number of nodules was determined.

The following compounds were tested: PGE2 (Cayman Chemical, Ann Arbor, MI); 17-phenyl-omega -trinor PGE2 (an EP1 agonist, Refs. 4 and 48, Cayman); butaprost (an EP2 agonist, Refs. 7 and 12, gift of Dr. P. Gardiner, Bayer, UK); sulprostone (an EP3/EP1 agonist, Refs. 2 and 7, gift of Dr. F. McDonald, Schering, Germany); 11-deoxy-PGE1 (an EP4/EP2 agonist, Ref. 48, Cayman); AH-23848B (an EP4 antagonist, Ref. 13, gift of Dr. S. Lister, Glaxo, UK); forskolin (an adenylate cyclase stimulator, ICN Biomedicals, Costa Mesa, CA); and DDA (2',3'-dideoxyadenosine, an adenylate cyclase inhibitor, Sigma).

Except for the experiment in which PGE2 was added to the culture medium for varying lengths of time (see RESULTS), all compounds that were tested were added to the cultures throughout the experimental period (days 0-21 for nodules and days 0-12 for AP) and were thus replaced with the medium twice weekly. Each compound was tested on 18 wells derived from six rats × triplicate repeats.

In addition to mineralized nodule formation, osteogenic differentiation was assessed by measuring AP activity in culture (27, 55). Femoral cells were cultured as before, and on day 12, they were washed in PBS and scraped in 10 mM Tris · HCl buffer (pH = 7.6) containing 10 mM MgCl2 and 0.1% Triton X-100. AP activity was determined colorimetrically with a Sigma kit on the basis of p-nitrophenylphosphate as substrate. The protein content was measured according to Bradford with BSA as standard and a protein assay kit (Bio-Rad, Munich, Germany), and enzyme activity was expressed as units per milligram protein.

All data are presented as means ± SE. Comparison between group means was performed with one-way analysis of variance with post hoc multi-group contrasts (n = 6 animals/group).


    RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

First, we determined the concentration range in which PGE2 was anabolic and added it at concentrations of 10-1,000 nM for 21 days. PGE2 increased bone nodule formation with a maximal effect at 100 nM (Figs. 1 and 2). This concentration was also maximally effective in stimulating AP activity (corrected for the protein content) in these cultures (Fig. 3). It is noteworthy that PGE2 repeatedly increased the protein content (by 10-20% at 6-12 days) but increased AP activity to a much greater degree.


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  		Fig. 1.   Representative wells showing mineralized and nonmineralized nodules after Von Kossa staining (×0.75). Cultures treated with prostaglandin E2 (PGE2; 100 nM) show increased number of mineralized nodules.


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  		Fig. 2.   PGE2 increases the number of mineralized nodules in bone marrow cultures, dose response. ** P < 0.01 vs. control, i.e., PGE2 = 0.


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  		Fig. 3.   PGE2 stimulates alkaline phosphatase (AP) activity in bone marrow cultures, dose response. ** P < 0.01 vs. control.

In search of the signal transduction involved in the anabolic effect of PGE2, we tested whether it is mediated via increased cAMP production. Indeed, the increase in nodule formation caused by PGE2 was mimicked by forskolin, an adenylate cyclase stimulator with maximal effect at 10 µM, and was blocked by DDA, an adenylate cyclase inhibitor (Fig. 4). These data pointed to EP2 and/or EP4 as the receptor mediating the effect of PGE2.


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  		Fig. 4.   Role of cAMP: increased nodule formation by PGE2 and forskolin (an adenylate cyclase stimulator) and inhibition of PGE2 effect by 2',3'-dideoxyadenosine (DDA, an adenylate cyclase inhibitor). * P < 0.05, ** P < 0.01 vs. control, i.e., PGE2 = 0.

In agreement with this finding, 17-phenyl-omega -trinor PGE2, an EP1 agonist, and sulprostone, an EP3/EP1 agonist, failed to increase nodule formation even at a concentration 10-fold higher than the most effective concentration of PGE2 (Fig. 5) and failed to stimulate AP activity (Fig. 6). When testing agonists of the cAMP stimulatory receptors (EP2 and EP4), we found that butaprost, a selective EP2 agonist, was ineffective in enhancing nodule formation, whereas 11-deoxy-PGE1, an EP4/EP2 agonist, was as effective as PGE2 in this assay, with maximal effect at 100 nM (Fig. 7). Similarly, 11-deoxy-PGE1, but not butaprost, stimulated AP activity, with maximal effect at 100 nM (Fig. 8). These data suggested that the anabolic effect of PGE2 in the bone marrow culture system was mediated via the EP4 receptor subtype. To validate this conclusion, we added to the cultures increasing concentrations of the selective EP4 antagonist AH-23848B in the presence of 100 nM PGE2. Both the increased nodule formation (Fig. 9) and enhanced AP activity (Fig. 10) caused by PGE2 were inhibited dose dependently by this compound down to the control group level, indicating the involvement of EP4 in the anabolic effect of PGE2.


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  		Fig. 5.   Lack of effect of EP1 agonist (17-phenyl-omega -trinor PGE2) and EP3/EP1 agonist (sulprostone) on mineralized nodule formation in bone marrow cultures. Effect of PGE2 serves as a positive control. ** P < 0.01 vs. control.


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  		Fig. 6.   Lack of effect of EP1 agonist (17-phenyl-omega -trinor PGE2) and EP3/EP1 agonist (sulprostone) on AP activity in bone marrow cultures. Effect of PGE2 serves as a positive control. * P < 0.05 vs. control.


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  		Fig. 7.   Stimulation of mineralized nodule formation in bone marrow cultures by EP4/EP2 agonist (11-deoxy-PGE1) but not EP2 agonist (butaprost). * P < 0.05, ** P < 0.01 vs. control.


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  		Fig. 8.   Stimulation of AP activity in bone marrow cultures by EP4 /EP2 agonist (11-deoxy-PGE1) but not EP2 agonist (butaprost). * P < 0.05, ** P < 0.01 vs. control.


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  		Fig. 9.   Stimulation of mineralized nodule formation in bone marrow cultures by 11-deoxy-PGE1 (an EP4/EP2 agonist) and inhibition of PGE2 stimulation by AH-23848B (an EP4 antagonist). * P < 0.05 vs. control.


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  		Fig. 10.   Inhibition of PGE2-stimulated AP activity in bone marrow cultures by AH-23848B (an EP4 antagonist). ** P < 0.01 vs. control.

All agents that increased the number of mineralized nodules also increased their proportion out of the total number of nodules (data not shown), suggesting that PGE2 recruits only osteogenic precursors to the adherent fraction of the culture. In support of this conclusion, we found that the correlation coefficient between the total number of nodules and the number of mineralized nodules in vehicle- and PGE2-treated cultures was extremely high (0.967), with a unity regression slope (Fig. 11). In all the experiments described, we counted the number of cells seeded on day 0 within each experiment and never found any significant difference in cell number among the wells subjected to different treatments. Furthermore, we attempted a correlation analysis between the number of mineralized nodules (as a dependent variable) and the total number of cells seeded and never found any such correlation. These observations indicate that the number of mineralized nodules was affected by the compounds added to the cultures and not by the number of cells seeded.


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  		Fig. 11.   Regression analysis between total number of nodules/well and number of mineralized nodules/well in control (PGE2 = 0, ) and PGE2-treated bullet  bone marrow cultures.

To examine the crucial period for the anabolic effect of PGE2 in this culture system, we added it in different time schedules to the culture medium. Addition of 100 nM PGE2 for the first 24 h only was as effective as the full 21 days in stimulating nodule formation (Fig. 12). Likewise, 24 h of PGE2 were equal to the full 12 days in stimulating AP activity (Fig. 13). These data indicated that the initial attachment period of 24 h is crucial for the stimulatory action of PGE2.


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  		Fig. 12.   Effect of time schedule of adding 100 nM PGE2 to cultures on mineralized nodule formation. ** P < 0.01 vs. control.


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  		Fig. 13.   Stimulation of AP activity measured on day 12 by PGE2 present for 12 days or for the 1st day only. * P < 0.05 vs. control.

Cumulatively, our data indicate that PGE2 increases the osteogenic capacity of bone marrow by recruiting osteoprogenitor cells via activation of the EP4 receptor subtype and that this effect probably occurs during the initial attachment period.


    DISCUSSION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

As reported previously by others (44, 46), PGE2 was anabolic in this bone marrow culture system, i.e., it increased the number of bone nodules and stimulated AP activity, with maximal effect at 100 nM. A similar anabolic activity was shown in a parallel system, fetal rat calvarial cells (15, 35, 50). In both systems, it is believed that each nodule originates from a single cell (fibroblastic colony-forming units), and therefore it was concluded that PGE2 recruits otherwise noncommitted osteogenic precursors. The recruitment of bone marrow osteogenic precursors in vitro is highly compatible with the proposed mode in which PGE2 enhances bone formation in vivo. Numerous animal experiments showed that both local and systemic administration of PGE2 augments bone mass by increasing the number of osteoblasts and the extent of bone forming surfaces and frequently by causing the formation of new bone tissue (3, 19, 20-23, 31-33, 52, 58). When the new bone formed was cancellous or endocortical, these new osteoblasts must have originated from bone marrow progenitors, which belong to the stromal compartment. In support of this mechanism whereby PGE2 stimulates bone formation, we recently showed that systemic administration of PGE2 to young rats induced the expression of early-response genes, such as c-fos and egr-1, in bone marrow cells and increased the size of the osteoprogenitor pool in bone marrow (54, 55). Cumulatively, these data show that osteoblast recruitment from marrow precursors is the major component of the anabolic action of PGE2 both in vivo and in vitro. Whether this recruitment involves proliferation or merely differentiation remains to be investigated.

We found that the highest concentration of PGE2 (1,000 nM) was not as stimulatory as the maximally effective one (100 nM). A similar observation was made by others in cultures of newborn-fetal rat calvaria cells (15, 35, 50). Whether this is due to some toxic effect that sets in under this concentration is not known; however, we noted that the increase in the protein content exerted by PGE2 (see RESULTS) was also not maximal (compared with 100 nM PGE2).

The anabolic effect of PGE2 in our marrow cultures was dependent on the concentration of Dex. We found that PGE2 increased the number of bone nodules only at a lower Dex concentration (10 nM) but not at a higher concentration (100 nM), which by itself increased nodule formation (data not shown). Similar observations made by Scutt et al. (45) showed that the stimulatory effect of 100 nM PGE2 on the number of calcified nodules, which was maximal at Dex concentrations of 1-10 nM, was greatly diminished at higher Dex concentrations. Dex is known to stimulate the differentiation of osteoblastic lineage cells in fetal rat calvaria cells (6), bone marrow cells (11, 30, 43), and other bone-related cell systems. In fact, cultures of bone marrow cells from adult rats grown without Dex do not form calcified nodules (30, 45). These observations establish that physiological concentrations of Dex are required for osteoblast differentiation in these culture systems and also permit the stimulatory (additive) effect of PGE2 as seen in our study too. Our data suggest that when the effect of Dex in recruiting marrow osteoprogenitors is maximal, PGE2 is no longer able to further enhance osteoblastic commitment. At higher, pharmacological doses, both effects of Dex are lost.

The exact mechanism whereby PGE2 recruits osteogenic precursors is not known. We found that the presence of PGE2 for the initial attachment period of 24 h is sufficient to induce the same increase in nodule formation as its presence for the full 21 days. A somewhat similar observation was made in bone marrow cultures (44) and in cultures of fetal rat calvarial cells (50) by others. Because each nodule is believed to originate from a single cell, these findings suggest that PGE2 induces, during the attachment period, a shift from nonadherent (noncommitted) to adherent (committed) marrow osteogenic colony-forming units. In support of this conclusion, we found in this study that treatment with PGE2 adds to the cultures only mineralized colonies, suggesting that it specifically affects inducible osteogenic colony-forming units.

We present significant evidence in this study that indicates that the anabolic effect of PGE2 in our system is mediated via the EP4 receptor subtype. First, this effect was mimicked by forskolin, an adenylate cyclase stimulator, and was inhibited by DDA, an adenylate cyclase inhibitor, pointing to EP2 and/or EP4 (the 2 cAMP-related receptors) as mediators. In agreement with this conclusion, 17-phenyl-omega -trinor PGE2 and sulprostone, agonists of the non-cAMP-related receptors (EP1 and EP3, respectively), were inactive. In support for a cAMP-dependent anabolic effect in bone cells, PGE2 and forskolin, but not sulprostone, were found to increase incorporation of [3H]thymidine and collagen synthesis in fetal rat calvaria organ cultures (56). Second, butaprost, a selective EP2 agonist, was ineffective in stimulating bone nodule formation and AP activity in our cultures, whereas 11-deoxy-PGE1 was as effective as PGE2. Butaprost is ~10-fold weaker than PGE2 at the EP2 receptor (7, 9, 28), but in our study even a concentration of butaprost 10-fold higher than that of PGE2 was ineffective. Although many of the agonists we tested in this study are not 100% selective for the respective EP receptors, they often have a 5- to 100-fold difference in their ability to activate the various EPs. For instance, 17-phenyl-omega -trinor PGE2 is equipotent relative to PGE2 at the EP1 receptor but ~5 times less active at the EP3 receptor and 50-100 times less active at the EP4 and EP2 receptors, respectively (9, 28). Conversely, sulprostone is equipotent relative to PGE2 at the EP3 receptor but ~2-4 times less active at the EP1 receptor. To date, there is no selective EP4 agonist; however, 11-deoxy-PGE1 binds the rat EP4 receptor with an affinity identical to that of PGE2 (9) and is used in conjunction with butaprost (a selective EP2 agonist) to distinguish the involvement of EP4 from that of EP2 (14, 28, 29, 34) as was done here. Thus our data so far implicated EP4 as the mediator of the anabolic effect of PGE2 in bone marrow of young adult rats. We further validated our conclusion by showing that this effect was gradually abolished by increasing concentrations of the weak but selective EP4 antagonist AH-23848B (13, 14). The possible involvement of EP4 in the recruitment of osteoblasts from bone marrow precursors is even further strengthened by our recent finding that EP4 is expressed in bone marrow of long bones of young adult rats such as the ones used for this study, whereas EP2 is not (Weinreb et al., unpublished data). However, unequivocal demonstration of the role of EP4 vs. EP2 in this assay must await the development of (yet unavailable) 100% selective EP2 and EP4 agonists that are equipotent relative to PGE2.

Our data corroborate those of Scutt et al. (46) that the anabolic effect of PGE2 in bone marrow cells is mediated via increased cAMP production. However, these authors have concluded that such an effect was consequently mediated via the EP2 receptor. The existence of EP4 was not known at that time and our present data, with various agonists and antagonists, point rather to EP4 as the mediator of this anabolic effect.

A recent study using RT-PCR showed that EP2 is expressed in fetal rat bones and that its expression is greatly diminished in young adult rats (38). These data, together with the report that butaprost partially stimulated proliferation and collagen synthesis in fetal rat calvariae (56), may point to a role for EP2 in fetal bone development and its possible replacement by EP4 in adult animals.

In summary, this study shows that PGE2 is anabolic in bone marrow cultures, i.e., increases the number of osteogenic colonies and enhances AP activity. This effect is apparently mediated by activation of the EP4 receptor subtype and occurs within the first 24 h, probably by recruiting noncommitted osteogenic precursors. The molecular cascade subsequent to the activation of EP4 in the noncommitted osteogenic precursors by PGE2 must be further investigated.


    ACKNOWLEDGEMENTS

This study was supported by the Lefcoe Fund for Oral Biology of the Goldschleger School of Dental Medicine and was carried out in the Rosenberg Bone Research Laboratory of the Goldschleger School of Dental Medicine.


    FOOTNOTES

This study was based on a Master of Science thesis submitted by N. Shir to Tel-Aviv University Goldschelger School of Dental Medicine.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: M. Weinreb, Dept. of Oral Biology, Maurice and Gabriela Goldschleger School of Dental Medicine, Tel-Aviv Univ., Tel-Aviv 69978, Israel.

Received 7 August 1998; accepted in final form 23 October 1998.


    REFERENCES
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References

1.   Abramovitz, M., M. Adam, Y. Boie, R. Grygorczyk, T. H. Rushmore, T. Nguyen, C. D. Funk, L. Bastien, N. Bawyer, C. Rochette, D. M. Slipetz, and K. M. Metters. Human prostanoid receptors: cloning and characterization. Adv. Prostaglandin Thromboxane Leukot. Res. 23: 499-504, 1995[Medline].

2.   Adam, M., Y. Boie, T. H. Rushmore, G. Muller, L. Bastien, K. T. McKee, K. M. Metters, and M. Abramovitz. Cloning and expression of three isoforms of human EP3 prostanoid receptor. FEBS Lett. 338: 170-174, 1994[Medline].

3.   Akamine, T., W. S. S. Jee, H. Z. Ke, X. J. Li, and B. Y. Lin. PGE2 prevents bone loss and adds extra bone to immobilized distal femoral metaphysis in female rats. Bone 13: 11-22, 1992[Medline].

4.   Armstrong, R. A., C. Marr, and R. L. Jones. Characterization of the EP receptor mediating dilatation and potentiation of inflammation in rabbit skin. Prostaglandins 49: 205-224, 1995[Medline].

5.   Bastien, L., N. Sawyer, R. Grygorczyk, K. M. Metters, and M. Adam. Cloning, functional expression and characterization of the human PGE2 receptor EP2 subtype. J. Biol. Chem. 269: 11873-11877, 1994[Abstract/Free Full Text].

6.   Bellows, C. J., J. E. Aubin, and J. N. M. Heersche. Physiological concentrations of glucocorticoids stimulate formation of bone nodules from isolated rat calvarial cells in vitro. Endocrinology 121: 1985-1992, 1987[Abstract].

7.   Bergmann, P., and A. Schoutens. Prostaglandins and bone. Bone 16: 485-488, 1995[Medline].

8.   Bernecker, P. M., L. G. Raisz, K. Nemoto, B. E. Kream, and C. C. Pilbeam. Effects of PGE2 and dexamethasone on primary mouse bone marrow cultures (Abstract). Bone 17: 558, 1995.

9.   Boie, Y., R. Stocco, N. Sawyer, D. M. Slipetz, M. D. Ungrin, F. Neuschafer-Rube, G. P. Puschel, K. M. Metters, and M. Abramovitz. Molecular cloning and characterization of the four rat prostaglandin E2 prostanoid receptor subtypes. Eur. J. Pharmacol. 340: 227-241, 1997[Medline].

10.   Centrella, M., S. Casinghino, and T. L. McCarthy. Differential actions of prostaglandins in separate cell populations from fetal rat bone. Endocrinology 135: 1611-1620, 1994[Abstract].

11.   Cheng, S. L., J. W. Yang, L. Rifas, S. F. Zhang, and L. V. Avioli. Differentiation of human bone marrow osteogenic stromal cells in vitro; induction of the osteoblast phenotype by dexamethasone. Endocrinology 134: 277-286, 1994[Abstract].

12.   Coleman, R. A., R. M. Eglen, R. L. Jones, S. Narumiya, T. Shimizu, W. L. Smith, S. E. Dahlen, J. M. Drazen, P. J. Gardiner, W. T. Jackson, T. R. Jones, R. D. Krell, and S. Nicosia. Prostanoid and leukotriene receptors: a progress report from the IUPHAR working parties on classification and nomenclature. Adv. Prostaglandin Thromboxane Leukot. Res. 23: 283-285, 1995[Medline].

13.   Coleman, R. A., S. P. Grix, S. A. Head, J. B. Louttit, A. Mallett, and R. L. G. Sheldrick. A novel inhibitory prostanoid receptor in piglet saphenous vein. Prostaglandins 47: 151-168, 1995.

14.   DeBrum-Fernandes, A. J., S. Morisset, G. Bkaily, and C. Patry. Characterization of the PGE2 receptor subtype in bovine chondrocytes in culture. Br. J. Pharmacol. 118: 1597-1604, 1996[Medline].

15.   Flanagan, A. M., and T. J. Chambers. Stimulation of bone nodule formation in vitro by prostaglandins E1 and E2. Endocrinolog y130: 443-448, 1992[Abstract].

16.   Foord, S. M., B. Marks, M. Stolz, E. Bufflier, N. J. Fraser, and M. G. Lee. The structure of the prostaglandin EP4 receptor gene and related pseudogenes. Genomics 35: 182-188, 1996[Medline].

17.   Friedenstein, A. J., I. I. Piatatzky-Shapiro, and K. V. Petrakova. Osteogenesis in transplants of bone marrow cells. J. Embryol. Exp. Morphol. 16: 381-385, 1966[Medline].

18.  Ikeda, T., C. Miyaura, A. Ichikawa, S. Narumiya, S. Yoshiki, and T. Suda. In situ localization of three subtypes (EP1, EP3 and EP4) of prostaglandin E receptors in embryonic and newborn mice (Abstract). J. Bone Min. Res. 10, Suppl. 1: S172, 1995.

19.   Ito, H., H. Z. Ke, W. S. S. Jee, and T. Sakou. Anabolic responses of an adult cancellous bone site to PGE2 in the rat. Bone Miner. 21: 219-236, 1993[Medline].

20.   Jee, W. S. S., H. Z. Ke, and X. J. Li. Long-term anabolic effects of PGE2 on tibial diaphyseal bone in male rats. Bone Miner. 15: 33-55, 1991[Medline].

21.   Jee, W. S. S., S. Mori, X. J. Li, and S. Chan. PGE2 enhances cortical bone mass and activates intracortical bone remodeling in intact and ovariectomized female rats. Bone 11: 253-266, 1990[Medline].

22.   Jee, W. S. S., K. Ueno, Y. P. Deng, and D. M. Woodbury. The effects of PGE2 in growing rats: increased metaphyseal hard tissue and cortico-endosteal bone formation. Calcif. Tissue Int. 37: 148-157, 1985[Medline].

23.   Jee, W. S. S., K. Ueno, D. B. Kimmel, D. M. Woodbury, P. Price, and L. A. Woodbury. The role of bone cells in increasing metaphyseal hard tissue on rapidly growing rats treated with PGE2. Bone 8: 171-178, 1987[Medline].

24.   Jorgensen, H. R. I., H. Svanholm, and A. Host. Bone formation induced in an infant by systemic PGE2 administration. Acta Orthop. Scand. 59: 464-466, 1988[Medline].

25.   Kasugai, S., S. Oida, T. Iimura, N. Arai, K. Takeda, K. Ohya, and S. Sasaki. Expression of PGE2 receptor subtypes in bone: expression of EP2 in bone development. Bone 17: 1-4, 1995[Medline].

26.   Katsuyama, M., N. Nishigaki, Y. Sugimoto, K. Morimoto, M. Negishi, S. Narumyia, and A. Ichikawa. The mouse prostaglandin E receptor EP2 subtype: cloning, expression and Northern blot analysis. FEBS Lett. 372: 151-156, 1995[Medline].

27.   Keila, S., S. Pitaru, A. Grosskopf, and M. Weinreb. Bone marrow from mechanically unloaded rat bones expresses reduced osteogenic capacity in vitro. J. Bone Miner. Res. 9: 321-327, 1994[Medline].

28.   Kiriyama, M., F. Ushikubi, T. Kobayashi, M. Hirata, Y. Sugimoto, and S. Narumiya. Ligand binding specificities of the eight types and subtypes of the mouse prostanoid receptors expressed in Chinese hamster ovary cells. Br. J. Pharmacol. 122: 217-224, 1997[Medline].

29.   Lydford, S. J., K. C. McKechnie, and I. G. Dougall. Pharmacological studies on prostanoid receptors in the rabbit isolated saphenous vein: a comparison with the rabbit isolated ear artery. Br. J. Pharmacol. 117: 13-20, 1996[Medline].

30.   Maniatopoulos, C., J. Sodek, and A. H. Melcher. Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res. 254: 317-330, 1988[Medline].

31.   Miller, S. C., and S. C. Marks. Local stimulation of new bone formation by PGE1: quantitative histomorphometry and comparison of delivery by minipumps and controlled-release pellets. Bone 14: 143-151, 1993[Medline].

32.   Mori, S., W. S. S. Jee, and X. J. Li. Production of new trabecular bone in osteopenic ovariectomized rats by PGE2. Calcif. Tissue Int. 50: 80-87, 1992[Medline].

33.   Mori, S., W. S. S. Jee, X. J. Li, S. Chan, and D. B. Kimmel. Effects of prostaglandin E2 on production of new cancellous bone in the axial skeleton of ovariectomized rats. Bone 11: 103-113, 1990[Medline].

34.   Mukhopadhyay, P., T. E. Geoghegan, R. V. Patil, P. Bhattacherjee, and C. A. Paterson. Detection of EP2, EP4 and FP receptors in human ciliary epithelial and ciliary muscle cells. Biochem. Pharmacol. 53: 1249-1255, 1997[Medline].

35.   Nagata, T., K. Kaho, S. Nishikawa, H. Shinohara, Y. Wakano, and H. Ishida. Effect of PGE2 on mineralization of bone nodules formed by fetal rat calvarial cells. Calcif. Tissue Int. 55: 451-457, 1994[Medline].

36.   Narumiya, S. Structures, properties and distributions of prostanoid receptors. Adv. Prostaglandin Thromboxane Leukot. Res. 23: 17-22, 1995[Medline].

37.   Negishi, M., Y. Sugimoto, and A. Ichikawa. Molecular mechanisms of diverse actions of prostanoid receptors. Biochim. Biophys. Acta 1259: 109-120, 1995[Medline].

38.   Nemoto, K., C. C. Pilbeam, S. R. Bilak, and L. G. Raisz. Molecular cloning and expression of a rat prostaglandin E2 receptor of the EP2 subtype. Prostaglandins 54: 713-725, 1997[Medline].

39.   Nishigaki, N., M. Negishi, A. Honda, Y. Sugimoto, T. Namba, S. Narumiya, and A. Ichikawa. Identification of prostaglandin E receptor `EP2' cloned from mastocytoma cells as EP4 subtype. FEBS Lett. 364: 339-341, 1995[Medline].

40.   Owen, M., J. Cave, and C. J. Joyner. Clonal analysis in vitro of osteogenic differentiation of marrow CFU-F. J. Cell Sci. 87: 731-738, 1987[Abstract/Free Full Text].

41.   Partridge, N. C., D. M. Alcorn, V. P. Michelangeli, B. E. Kemp, G. B. Ryan, and T. J. Martin. Functional properties of hormonally responsive cultured normal and malignant rat osteoblastic cells. Endocrinology 108: 213-219, 1981[Abstract].

42.  Raisz, L. G., C. C. Pilbeam, and P. M. Fall. Prostaglandins: mechanisms of action and regulation of production in bone. Osteoporosis Int. 3, Suppl. 1: S136-S140, 1993.

43.   Rickard, D. J., T. A. Sullivan, B. J. Shenker, P. S. Leboy, and I. Kazhdan. Induction of rapid osteoblast differentiation in rat bone marrow stromal cell cultures by dexamethasone and BMP-2. Dev. Biol. 161: 218-228, 1994[Medline].

44.   Scutt, A., and P. Bertram. Bone marrow cells are targets for the anabolic actions of PGE2 on bone: induction of a transition from non-adherent to adherent osteoblast precursors. J. Bone Miner. Res. 10: 474-487, 1995[Medline].

45.   Scutt, A., P. Bertram, and M. Brautigam. The role of glucocorticoids and PGE2 in the recruitment of bone marrow mesenchymal cells to the osteoblastic lineage: positive and negative effects. Calcif. Tissue Int. 59: 154-162, 1996[Medline].

46.   Scutt, A., M. Zeschnigk, and P. Bertram. PGE2 induces the transition from non-adherent to adherent bone marrow mesenchymal precursor cells via a cAMP/EP2-mediated mechanism. Prostaglandins 49: 383-395, 1995[Medline].

47.  Smock, S. L., S. P. McCurdy, L. C. Pan, and T. A. Owen. Regulation of alkaline phosphatase and IGF-1 by PGE2 in bone marrow cells (Abstract). J. Bone Miner. Res. 11, Suppl. 1: S177, 1996.

48.  Suda, M., K. Tanaka, K. Natsui, C. Shigeno, J. Konishi, S. Narumiya, and K. Nakao. Cell growth and differentiation are mediated by different subtypes of prostaglandin E receptor in osteoblastic cell line (Abstract). J. Bone Miner. Res. 10, Suppl. 1: S248, 1995.

49.   Suponitzki, I., and M. Weinreb. Differential anabolic effects of PGE2 in long bones and calvariae of young rats. J. Endocrinol. 156: 51-57, 1998[Abstract].

50.   Tang, L. Y., D. B. Kimmel, W. S. S. Jee, and J. A. Yee. Functional characterization of PGE2 inducible osteogenic CFU in cultures of cells isolated from the neonatal rat calvarium. J. Cell. Physiol. 166: 76-83, 1996[Medline].

51.   Ueda, K., A. Saito, H. Nakano, M. Aoshima, M. Yokota, R. Muraoka, and T. Iwaya. Cortical hyperostosis following long-term administration of PGE1 in infants with cyanotic congenital heart disease. J. Pediatr. 97: 834-836, 1980[Medline].

52.   Ueno, K., T. Haba, D. Woodbury, P. Price, R. Anderson, and W. S. S. Jee. The effects of prostaglandin E2 in rapidly growing rats: depressed longitudinal and radial growth and increased metaphyseal hard tissue mass. Bone 6: 79-86, 1985[Medline].

53.   Watabe, A., Y. Sugimoto, A. Honda, A. Irie, T. Namba, M. Negishi, S. Ito, S. Narumiya, and A. Ichikawa. Cloning and expression of cDNA for a mouse EP1 subtype of prostaglandin E receptor. J. Biol. Chem. 268: 20175-20178, 1993[Abstract/Free Full Text].

54.   Weinreb, M., S. J. Rutledge, and G. A. Rodan. Systemic administration of an anabolic dose of PGE2 induces early-response genes in rat bones. Bone 20: 347-353, 1997[Medline].

55.   Weinreb, M., I. Suponitzky, and S. Keila. Systemic administration of an anabolic dose of PGE2 in young rats increases the osteogenic capacity of bone marrow. Bone 20: 521-526, 1997[Medline].

56.   Woodiel, F. N., P. M. Fall, and L. G. Raisz. Anabolic effects of prostaglandins in cultured fetal rat calvariae: structure-activity relations and signal transduction pathways. J. Bone Miner. Res. 11: 1249-1255, 1996[Medline].

57.   Yamaguchi, D. T., T. J. Hahn, T. G. Beeker, C. R. Kleeman, and S. Muallem. Relationship of cAMP and calcium messenger systems in prostaglandin-stimulated UMP-106 cells. J. Biol. Chem. 263: 10745-10753, 1988[Abstract/Free Full Text].

58.   Yang, R. S., T. K. Liu, and S. Y. Lin-Shiau. Increased bone growth by local PGE2 in rats. Calcif. Tissue Int. 52: 57-61, 1993[Medline]. thesis

Am J Physiol Endocrinol Metab 276(2):E376-E383
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