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Department of Medicine (X.L., Y.O., C.C.P., J.A.L., L.G.R.), University of Connecticut Health Center, Farmington, Connecticut 06030; and Department of Medicine (C.R.J.K., R.M.B.), Vanderbilt University Medical Center, Nashville, Tennessee 37232
Address all correspondence and requests for reprints to: Lawrence G. Raisz, M.D., Division of Endocrinology/Metabolism MC1850, University of Connecticut Health Center, Farmington, Connecticut 06030. E-mail: raisz{at}nso.uchc.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Osteoclast-like cell formation, as determined by tartrate-resistant acid phosphatase-positive multinucleated cells (TRAP + MNC) in bone marrow cultures and cocultures of spleen and osteoblastic cells, has been employed to study osteoclastogenesis since the 1980s (9, 10, 11). Receptor activator of nuclear factor (NF)-kB ligand (RANKL), also called TNF-related activation induced cytokine (TRANCE), osteoclast differentiation factor (ODF) or osteoprotegerin ligand (OPGL) is clearly necessary for osteoclast formation (12, 13). In addition, macrophage colony stimulating factor (M-CSF) is essential, but not sufficient, for osteoclast formation (14, 15). A combination of RANKL and M-CSF stimulates osteoclast formation in spleen cell cultures (16). There are also factors that can inhibit osteoclast formation. Osteoprotegerin (OPG) is a decoy receptor for RANKL, which blocks its interaction with RANK (17, 18). Granulocyte/macrophage colony stimulating factor (GM-CSF) is also a potent inhibitor of osteoclast formation in the mouse system (19, 20).
PGE2 stimulates osteoclast formation in bone marrow cultures (10, 21), increases expression of messenger RNA for RANKL, and decreases OPG messenger RNA (mRNA) expression in osteoblastic cells (13, 22). While these studies suggested that PGE2 acts on osteoblastic cells to stimulate osteoclast formation, a recent study suggested that PGE2 may also act synergistically with RANKL and M-CSF on hematopoietic precursors to induce TRAP+ MNC (23). PGE2 inhibits GM-CSF production in bovine lymphocytes (24).
In the present study, we compared the ability of EP2 wild-type (EP2 +/+) and knockout (EP2 -/-) mice to form TRAP + MNC in cultures of murine bone marrow, in cocultures of spleen cells and osteoblast-like cells, and in spleen cell cultures stimulated with PGE2 in the presence of RANKL and M-CSF. RANKL and OPG mRNA expression in osteoblastic and bone marrow cell cultures and GM-CSF mRNA expression in spleen cell cultures were also examined. We found that cultures from EP2 -/- mice showed a decreased osteoclastogenic response not only to PGE2 but also to PTH and 1,25-dihydroxyvitamin D [1,25-(OH)2D3]. The major defect appeared to be in the cells of osteoblastic lineage, which support osteoclast differentiation. There also appeared to be an additional defect in the response of the cells of hematopoietic lineage, which form osteoclasts.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials
Alpha MEM (
-MEM) and FCS were purchased from Life Technologies, Inc. (Grand Island, NY).
PGE2 and PTH (PTH, 1–34) were purchased from
Sigma (St. Louis, MO).
1,25(OH)2D3 was obtained
from BIOMOL Research Laboratories, Inc. (Plymouth Meeting,
PA). RANKL was a gift from Dr. Dirk Anderson (Immunex Corp., Seattle, WA). M-CSF, murine granulocyte macrophage-colony
stimulating factor (GM-CSF), polyclonal antibody to GM-CSF and IgG were
obtained from R&D systems (Minneapolis, MN). The compound L-161982, a
selective EP4 receptor antagonist
(EP4RA), was kindly provided by Dr. R. N.
Young (Merck Frosst Canada Inc.). This compound blocks
PGE2 binding to EP4
receptors with IC50 of 30 nM and shows minimal inhibition
of the EP2 receptor binding (IC50, 58
µM) (25). EP4RA inhibited
PGE2 stimulated intracellular cAMP by 44% in
osteoblastic cell cultures (unpublished data, Tomita, M., X. Li,
and L. G. Raisz).
Genotyping of mice
When the mice were 3–4 weeks old, ears were punched or notched
for identification. Tail samples were obtained for analysis by PCR.
Primer sequences were as follow: mEP2.249s:
5'-ccg ggg ttc tgg gga atc-3'; pPNT.1803s: 5'-ttg cca agt tct aat tcc
atc aga-3'; and mEP2.801a: 5'-cat gcg aat gag gtt
gag gat aa-3'. mEP2.249s and pPNT.1803s amplify
the wild-type gene and give a 575-bp single band; pPNT.1803s and
mEP2.801a amplify the recombinant gene and give a
295 bp single band. The conditions for PCR of the
EP2 receptor were 1 cycle for 1 min at 95 C; 35
cycles for 30 s at 95 C, 30 s at 56 C, and 1 min at 72 C.
Products were electrophoresed on a 2% agarose gel in 1 x TAE
buffer at 100 V. Positive controls for each genotype were included in
each amplification, as was a negative control (no DNA).
Bone marrow cell cultures
Mouse bone marrow cells were isolated by a modification of
previously published methods (9, 10). Most experiments were done when
the mice were 5–6 weeks old. However, in some experiments the mice
were 3–4 months old. Bone ends of aseptically dissected tibiae were
cut off with scissors, and the marrow cavities were flushed with 1 ml
-MEM. The collected marrow cells were washed twice with -MEM and
cultured in 0.5 ml of -MEM containing 10% FCS at 1 x
106 cells/well in 24-well plates
(Costar, Corning, NY). The cultures were fed every 3 days
by replacing 0.4 ml of old medium with fresh medium. Osteoclast-like
cell formation, which was determined as TRAP + MNC formation, was
evaluated in the presence or absence of PGE2 (1
µM), 1,
25(OH)2D3 (10
nM), PTH (10 nM), or
EP4RA (1 µM). All cultures were
maintained at 37 C in a humidified atmosphere of 5% CO2 in air. After
being cultured for 7 days, the cells were washed with PBS and fixed
with 2.5% glutaraldehyde in PBS for 30 min at room temperature. TRAP
staining was performed with a commercial kit (leukocyte acid
phosphatase kit, Sigma). TRAP + MNC per well were counted
in six separate wells for each condition of culture.
Spleen and calvarial osteoblastic cell cocultures
Mouse osteoblastic cells were obtained by sequential collagenase
digestion (26). Briefly, calvariae from 5-week-old mice were dissected
free of loosely adherent fibrous tissue. Calvariae were then washed in
PBS and incubated at room temperature with gentle shaking for five
sequential 20 min digestions, each with 4 ml of 0.1% bacterial
collagenase (Collagenase P, Roche Molecular Biochemicals,
Indianapolis, IN), 0.05% trypsin, and 4 mM Na2 EDTA in
Ca2+, Mg2+ free PBS. After
each digestion, calvariae were placed in fresh digestion solution, and
the liberated cells were collected by centrifugation and washed three
times in 3 ml of -MEM with 10% heat-inactivated FCS (Life Technologies, Inc.). Cell populations harvested from the second
to the fifth digestions were combined and cultured to confluence in
-MEM with 10% FCS in 100-mm plates.
Spleens from 6-week-old EP2 +/+ or
EP2 -/- mice were processed to produce a
suspension of spleen cells in -MEM containing 10% FCS as previously
described (16). Spleen cells (1 x 106
cells/well) were co-cultured with osteoblastic cells (1 x
104 cells/well) in 0.5 ml -MEM supplemented
with 10% FCS in 24-well plates as described previously (11). Cultures
were maintained for 7 days following the same protocol described for
bone marrow cultures. Osteoclast formation estimated by TRAP + MNC
formation in the coculture system was evaluated in the presence or
absence of PGE2 (1 µM) and PTH (10
nM). At the end of the culture period, the cells were
stained for TRAP and TRAP + MNC per well were counted in six separate
wells for each culture condition.
Spleen cell cultures
Spleens from EP2 +/+ and
EP2 -/- mice (6 weeks old) were processed as
described above. Spleen cells (1 x 106
cells/well) were cultured in 0.5 ml -MEM supplemented with 10% FCS
in 24-well plates both in the presence and absence of M-CSF (10 ng/ml)
and RANKL (10 ng/ml). The RANKL and M-CSF-treated spleen cultures were
further treated with or without PGE2 (1
µM) and a selective EP4RA (1
µM). In all cultures, medium and added factors were
replaced every 3 days. On day 7 of culture, the cells were stained and
TRAP+ MNC were counted. RANKL and M-CSF-treated spleen cultures were
also treated with GM-CSF (1 ng/ml) with or without
PGE2 (1 µM), a polyclonal antibody
(1 µg/ml) to GM-CSF (Anti-GM-CSF) or IgG (1 µg/ml). TRAP+ MNC
formation was measured as above.
Pit formation assay
Bone resorption was assayed by measuring the ability of cultured
bone marrow cells to form resorption pits on devitalized bovine
cortical bone slices (4.4 x 4.4 x 0.2 mm) using previously
described methods (27). Briefly, trypsinized bone marrow cells that had
been cultured for 7 days with or without PGE2
were transferred onto the surface of bone slices for 90 min in PBS.
Bone slices were rinsed vigorously and incubated for 24 h at 37 C
in -MEM (0.7 g of sodium bicarbonate per liter) with 10% HIFCS.
After incubation, bone slices were stained with 1% toluidine blue in
1% borax. The number of resorption pits was counted using reflected
light microscopy.
Northern blot analysis
The osteoblastic cells of EP2 +/+ and
EP2 -/- mice were obtained as described above.
After 6-days of culture, the cells were treated with
PGE2 (1 µM) or vehicle for 24
h and RNA was extracted by the acid guanidine isothiocyanate method
(28, 29). Bone marrow cells were cultured with
PGE2 (1 µM) or vehicle for 7 days,
and total RNA was extracted by the same method.
After quantitation at 260 nm, 20 µg of RNA was run on a 1% agrose-2.2 M formaldehyde gel, transferred to a nylon membrane (GeneScreen plus; NEN Life Science Products, Boston, MA) by Turboblotter (Schleicher & Schuell, Keene, NH) and fixed to the membrane by UV irradiation (Stratolinker; Stratagene). After 3 h of prehybridization in 50% formamide solution at 42 C, filters were hybridized overnight at 42 C in a similar solution with random primer [32P] dCTP-labeled complementary DNA probe for RANKL, OPG or G3PDH. [32P] dCTP was from NEN Life Science Products. Filters were washed once in a 1 x SSC, 1% SDS solution at room temperature, once in a 0.1 x SSC, 0.1% SDS solution at 65 C, and then three more times in the latter solution at room temperature. After washing, the filter was exposed to Kodak XAR-5 film at -70 C. Images were digitalized by scanner (ScanJet, Hewlett-Packard Co., Corvallis, OR) connected to a computer (Power Macintosh 7500/100), and optical density was determined using a digital image processing and analysis program (NIH Image 1.61, NIH, Bethesda, MD).
RNA extraction and reverse transcriptase PCR amplification for
spleen cell cultures
Spleen cells (5 x 106 cells/well)
were plated and maintained in 2 ml -MEM supplemented with 10% FCS
in 6-well plates both in the absence and presence of M-CSF (10 ng/ml)
and ODF (10 ng/ml). The RANKL and M-CSF-treated spleen cultures were
further treated with or without PGE2 (1
µM). RNA was extracted after a 7-day treatment. RT and
PCR were done as previously reported (29, 30). Briefly, 2 µg aliquots
of total RNA were reverse-transcribed using oligo (dT) as a primer (1.5
µM final concentration) in 30 µl of RT-solution. Five
microliters of RT-solution were then amplified.
EP2 receptor PCR. Primer sequences for EP2 receptor were as follow: forward mEP2 receptor: 5'-cca cca tgg act aca agg acg acg atg aca agg aca att ttc tta atg act c-3'; reverse mEP2 receptor: 5'-agc gca tcc tca caa ctg tc-3'. The conditions for PCR of the EP2 receptor were 30 cycles for 45 sec at 94 C, 45 sec at 57 C, and 2 min 15 sec at 72 C. Products were electrophoresed on a 1% agarose gel in 1 x TBE buffer at 100 V, stained with ethidium bromide and photographed under UV illumination. Positive controls for each genotype were included in each amplification, as was a negative control (no DNA). Images were captured by Alpha Imager 2200 Documentation & Analysis System (Alpha Innotech Co., San Leandro, CA). Optical density was determined as described above.
G3PDH PCR. Primer sequences for G3PDH were as follow: forward G3PDH: 5'-tga agg tcg gtg tga acg gat ttg gcc-3'; reverse G3PDH: 5'-cat gta ggc cat gag gtc cac cac-3'. The conditions for PCR of the G3PDH were 27 cycles for 45 sec at 94 C, 45 sec at 57 C, and 2 min 15 sec at 72 C. The amplified samples were run as described above. Images and optical density were processed as described above.
GM-CSF PCR. PCR amplification was done using gene-specific PCR primers and Taq polymerase (Life Technologies, Inc.). Specific amplifier sets for GM-CSF were purchased from CLONTECH Laboratories, Inc. (Palo Alto, CA). The amplification cycle consisted of denaturation at 94 C for 45 sec, annealing at 60 C for 45 sec, and extension at 72 C for 2 min for 35 cycles. After the last cycle, the mixture was incubated at 72 C for 7 min. The amplified samples were run as described above. Images and optical density were processed as described above.
Statistical analysis
The significance of the difference between two groups was
evaluated with unpaired two-tailed Student’s t test. The
comparisons among multiple groups were done by one-way ANOVA with
significance determined by Scheffé’s method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mouse bone marrow cell cultures
Treatment with PGE2 and EP4RA. There
were fewer than five TRAP+ MNC/well in vehicle-treated bone marrow
cultures from EP2 +/+ and
EP2 -/- mice. PGE2
increased TRAP+MNC in EP2 +/+ marrow cultures
(Fig. 1
).
PGE2-treated EP2 -/- bone
marrow cultures showed an 83–90% decrease in TRAP+ MNC compared with
PGE2-treated EP2 +/+ bone
marrow cultures in three different experiments (Table 1). Treatment of bone marrow cultures
with EP4RA reduced PGE2
stimulated TRAP+ MNC by 60% in cultures from EP2
+/+ mice and 73% in cultures from EP2 -/- mice
(Fig. 1).
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). Similarly, PTH-treated EP2
-/- bone marrow cultures showed a 44–73% decrease in TRAP+ MNC
formation compared with PTH-treated EP2 +/+ bone
marrow cultures in two different experiments.
Spleen and osteoblastic cell cocultures
Cocultures without stimulation. Unstimulated cultures produced
few TRAP + MNC and showed a significant (P < 0.05)
decrease when either spleen cells or osteoblasts in the co-cultures
were from EP2 -/- mice (Fig. 2).
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, Table 2). PGE2-treated
cocultures of EP2 -/- osteoblastic cells and
EP2 +/+ or EP2 -/- spleen
cells showed a marked decrease (88–92%) in TRAP+ MNC formation in
three different experiments. There were also 43 to 64% fewer TRAP+ MNC
when EP2 +/+ osteoblastic cells were co-cultured
with EP2 -/- spleen cells in three different
experiments (Fig. 2, Table 2).
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). PTH-treated cocultures of
EP2 -/- osteoblastic cells and
EP2 +/+ or EP2 -/- spleen
cells showed a marked decrease (68–85%) in TRAP+ MNC formation in two
different experiments. There were also 21–48% fewer TRAP+ MNC when
EP2 +/+ osteoblastic cells were cocultured with
EP2 -/- spleen cells in two different
experiments.
Spleen cell cultures
Cultured spleen cells treated with RANKL plus M-CSF from
EP2 +/+ mice expressed the
EP2 receptor (Fig. 3) and expression of
EP2 receptor mRNA decreased after 7 days of
treatment with PGE2. No EP2
receptor mRNA signal was obtained from EP2 -/-
cells.
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). PGE2 (1
µM) increased TRAP+ MNC by 2.5 fold in RANKL and
M-CSF-treated EP2 +/+ spleen cultures. However,
PGE2 had no effect on TRAP + MNC formation in
EP2 -/- spleen cell cultures. Furthermore,
EP4RA did not alter the number of TRAP + MNC that
formed in either EP2 +/+ or
EP2 -/- spleen cultures that were treated with
RANKL and M-CSF. In another experiment, PGE2 in
the presence of RANKL and M-CSF increased TRAP+ MNC 5-fold in
EP2 +/+ spleen cultures, while it had no effect
on the number of TRAP+ MNC that formed in EP2
-/- spleen cell cultures (data not shown).
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, PGE2-treated
cultures of EP2 +/+ bone marrow cells produced
numerous large pits (20.2 ± 0.5/slice) compared with 0.5 ±
0.3 pits/slice in control cultures. PGE2-treated
EP2 -/- cultures produced 2.0 ± 0.6
pits/slice compared with 0.8 ± 0.4 in control cultures. While
fewer pits were produced by PGE2-treated
EP2 -/- cultures, the pits that did form
were of similar appearance and size to those in cultures from
EP2 +/+ mice.
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) and bone marrow cell cultures (Fig. 6B) from EP2 +/+ and EP2
-/- mice. Control cultures of primary osteoblastic cells expressed
RANKL and OPG mRNA. PGE2 treatment increased
relative RANKL mRNA expression by 117% in osteoblastic cell cultures
of EP2 +/+ mice, but by only 34% in cultures of
EP2 -/- mice. There was little effect of
PGE2 on OPG mRNA expression in osteoblastic cell
cultures. In bone marrow cultures, 7 days of PGE2
treatment increased RANKL mRNA expression by 115% in
EP2 +/+ cultures, whereas there was only a 30%
increase in EP2 -/- cultures, as in
osteoblastic cells. PGE2 had little effect on OPG
mRNA expression in EP2 +/+ bone marrow cell
cultures.
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). GM-CSF treatment inhibited TRAP+ MNC
formation in EP2 +/+ cultures and blocked the
effect of PGE2 (Fig. 8). A neutralizing polyclonal antibody to
murine GM-CSF increased TRAP+ MNC in cultures treated with RANKL and
M-CSF and reversed the inhibitory effects of GM-CSF (Fig. 8) but did
not further increase the number of TRAP+ MNC in
PGE2-treated cultures (data not shown).
Nonspecific IgG did not reverse the inhibitory effects of GM-CSF.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The defect in the ability of osteoblastic cells from EP2 -/- mice to support osteoclastogenesis was demonstrated by an 88% decrease in osteoclast-like cell formation in PGE2-treated cultures and a 68% decreased in PTH-treated cultures when EP2 +/+ spleen cells were cultured with EP2 -/- osteoblastic cells compared with cocultures of EP2 +/+ spleen cells with EP2 +/+ osteoblastic cells. The defect in EP2 -/- osteoblast-like cells was associated with a decrease in the ability of PGE2 to increase RANKL, whereas the defect in EP2 -/- spleen cells was associated with a failure of PGE2 to decrease GM-CSF mRNA expression. Increased RANKL and decreased OPG mRNA expression have been demonstrated in response to stimulators of bone resorption (31). Changes in the balance between RANKL and OPG mRNA expression may be important in the role of PGs in regulating osteoclast formation. However, further studies of protein levels as well as exploration of other pathways are needed to confirm this hypothesis.
It is likely that the ability of PGE2 to increase TRAP + MNC number in spleen cell cultures treated with RANKL and M-CSF depends on the action of PGE2 to inhibit GM-CSF production. GM-CSF has previously been shown to inhibit osteoclastogenesis in bone marrow cultures, and it is the mediator of the inhibitory effect of IL-18 on osteoclast like cell formation (20). Moreover decreased GM-CSF production has previously been observed in bovine lymphocytes treated with PGE2 (24). In our culture system, GM-CSF blocked osteoclast-like cell formation. An antibody to GM-CSF reversed the effect of GM-CSF but did not further increase the response to PGE2. Recent experiments with cells from mice in which the inducible cyclooxygenase (COX-2) gene was knocked out confirm the effect of PGE2 on GM-CSF production (Okada, Y., and C. C. Pilbeam, unpublished data). It is possible that endogenous prostaglandins, produced by COX-2, may increase osteoclast number by diverting cells that might otherwise differentiate along the macrophage pathway toward the osteoclast lineage.
Stimulation of adenylyl cyclase by prostaglandins can be mediated not only by the EP2, but also by the EP4 receptor. Recent studies suggest that animals lacking a functional EP4 receptor also have a defect in osteoclast-like cell formation in bone marrow cultures and co-cultures of spleen and calvarial osteoblastic cells (32, 33). In our own studies, we have found that a recently developed EP4 receptor selective inhibitor can further reduce the number of TRAP+ MNC in marrow cultures from either EP2 +/+ or EP2 -/- mice. However, a role for EP4 has been demonstrated only in cells of the osteoblast lineage and not in the hematopoietic osteoclast precursors (32). Our results confirm this finding because we showed that EP4RA reduced osteoclast formation in bone marrow cultures but had no effect on the stimulation of osteoclast-like cell formation by PGE2 in spleen cell cultures that were treated with M-CSF and RANKL.
A role for prostaglandins in osteoclast production in response to other stimulators, including PTH, 1,25(OH)2D3, FGF-2 and IL-1, has been suggested by studies that use nonsteroidal antiinflammatory drugs to inhibit prostaglandin production (1, 27, 30, 34, 35, 36). Recent data from our laboratory indicate that endogenous prostaglandin production, which facilitates osteoclast-like cell formation, is dependent upon the presence of COX-2 (37). In contrast to these cell culture experiments, studies using organ cultures or in vivo models to demonstrate a role for endogenous prostaglandins in the resorptive response to different stimulators have produced variable results. One reason for this discrepancy may be that prostaglandins are most important in early replication and differentiation of the osteoclast precursors. In organ culture and in vivo, there may already be ample numbers of osteoclast precursors available for the final stages of osteoclast precursor differentiation, fusion and activation. Hence, the cell culture models that was employed in the present study may be more representative of the hyperstimulation of osteoclastogenesis that occurs in pathologic conditions like inflammation, rather than physiologic bone remodeling.
Despite these reservations, the identification of the roles of EP2, and also of EP4 receptors in mediating bone resorption should have important clinical implications. There is evidence that the bone loss associated with inflammation and immobilization is due to local production of prostaglandins (38, 39). Moreover, in rodent models, prostaglandins that are produced in response to increased cytokines may mediate the increased bone resorption observed after ovariectomy (29). Further studies in knockout animals and the development of specific agonists and antagonists for the EP2 and EP4 receptors should help define the roles of prostaglandins and their receptors in skeletal physiology and pathophysiology.
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Acknowledgments |
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Footnotes |
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Received October 29, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, TNF, basic FGF, TGFß in mice deficient in
EP4 subtype of PGE receptor. Bone 23:S215
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