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 |
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-
-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 |
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 |
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
-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
-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-
-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 |
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.
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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.
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In agreement with this finding, 17-phenyl-
-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- -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- -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.
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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 bone marrow
cultures.
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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.
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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 |
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-
-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-
-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.
 |
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