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J. Biol. Chem., Vol. 275, Issue 26, 19819-19823, June 30, 2000
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From the
Received for publication, March 13, 2000
Prostaglandin E2
(PGE2) acts as a potent stimulator of bone resorption. In
this study, we first clarified in normal ddy mice the involvement of
protein kinase A and induction of matrix metalloproteinases (MMPs) in
PGE2-induced bone resorption, and then identified PGE receptor subtype(s) mediating this PGE2 action using mice
lacking each subtype (EP1, EP2, EP3, and EP4) of PGE receptor. In
calvarial culture obtained from normal ddy mice, both PGE2
and dibutyryl cyclic AMP (Bt2cAMP) stimulated bone
resorption and induced MMPs including MMP-2 and MMP-13. Addition of an
inhibitor of protein kinase A, H89, or an inhibitor of MMPs, BB94,
significantly suppressed bone-resorbing activity induced by
PGE2. In calvarial culture from EP1-, EP2-, and
EP3-knockout mice, PGE2 stimulated bone resorption to an
extent similar to that found in calvaria from the wild-type mice. On
the other hand, a marked reduction in bone resorption to
PGE2 was found in the calvarial culture from EP4-knockout
mice. The impaired bone resorption to PGE2 was also
detected in long bone cultures from EP4-knockout mice.
Bt2cAMP greatly stimulated bone resorption similarly in
both wild-type and EP4-knockout mice. Induction of MMP-2 and MMP-13 by
PGE2 was greatly impaired in calvarial culture from
EP4-knockout mice, but Bt2cAMP stimulated MMPs induction
similarly in the wild-type and EP4-knockout mice. These findings
suggest that PGE2 stimulates bone resorption by a
cAMP-dependent mechanism via the EP4 receptor.
Prostaglandins (PGs)1
are produced in the bone mainly by osteoblasts and stimulate both bone
formation and resorption (1-3). Among several PGs produced,
PGE2 is a major product, and its production by osteoblasts
is regulated by several cytokines including interleukin-1 (IL-1). We
previously reported that cytosolic phospholipase A2 is
responsible for the release of arachidonic acid in osteoblasts, and
that the conversion of arachidonic acid to PGE2 is then
catalyzed by cyclooxygenase (COX)-2 induced in response to IL-1 (4). In vitro, PGE2 production primarily leads to
bone resorption (3, 5); PGE2 stimulates adenylate cyclase
in osteoblasts, induces osteoclast formation in co-cultures of bone
marrow cells and osteoblasts, and stimulates bone resorption in
calvarial cultures (5, 6). In contrast, PGE2 stimulates
both bone formation and resorption in vivo. The mechanism of
the anabolic effect of PGE2 still remains unclarified.
The actions of PGE2 are mediated by rhodopsin-type
receptors specific to this PG. There are four subtypes of PGE receptors designated as EP1, EP2, EP3, and EP4 that are encoded by different genes and expressed differently in each tissue (7-10). The
intracellular signaling also differs among the receptor subtypes; EP1
is coupled to Ca2+ mobilization, and EP3 inhibits adenylate
cyclase, whereas both EP2 and EP4 stimulate adenylate cyclase (7, 8).
To identify the physiological functions of each EP receptor subtype, we
have generated mice lacking respective receptors by homologous
recombination (11-13). Loss of EP4 is not lethal in utero,
but most EP4 ( We previously reported that cytokines with bone-resorbing activity such
as IL-1 and IL-6 greatly induce the expression of matrix
metalloproteinases (MMPs) including MMP-13 (type 3 collagenase) and
MMP-2 (gelatinase A) in mouse calvarial cultures, and that the potency
of various cytokines to induce MMPs is closely correlated to their
bone-resorbing activity that involves the degradation of bone matrix
(15). Using collagenase-resistant mutant mice, Zhao et al.
(16) recently reported that the cleavage of type I collagen by
collagenase such as MMP-13 is essential for the induction of
osteoclastic bone resorption. Therefore, monitoring the induction of
MMPs in mouse calvarial cultures appears to be a suitable measure for
the bone-resorbing activity.
In this study, we used EP1-, EP2-, EP3-, and EP4-knockout mice and
examined the mechanism of action of PGE2 in bone
resorption. We identified the EP4 subtype of PGE receptor responsible
for transducing signals for bone-resorbing activity of
PGE2.
Animals and Reagents--
Newborn mice of the ddy strain were
obtained from Japan SLC Inc. (Shizuoka, Japan). Mice lacking EP1, EP2,
EP3, and EP4 were generated, and homozygote, heterozygote and wild-type
mice of the F2 progeny were used (11-13). To examine the genotype of
each mouse, polymerase chain reaction analysis was performed on DNA extracted from the tail or brain of neonates, using the oligonucleotide primers designed to detect the respective EP locus and Neo cassette, as
reported previously (11-13). PGE2 and dibutyryl cyclic AMP
(Bt2cAMP) were obtained from Sigma. H89, an inhibitor of
protein kinase A, was purchased from Seikagaku Kogyo Co. (Tokyo,
Japan). BB94, an inhibitor of MMPs, was kindly donated by British
Biotech Pharmaceuticals Ltd. (Oxford, UK). All other chemicals were of
analytical grade.
Bone Resorption Assay--
One-day-old mice were killed, and
their calvariae, radii, and ulnae were aseptically isolated and
dissected free of suture tissues. For calvarial culture, the calvaria
were divided into halves and cultured for 24 h at 37° C under
5% CO2 in air in 0.12 ml of BGJb medium (Life
Technologies, Inc.) containing 1 mg/ml bovine serum albumin (fraction
V, Sigma). After pre-culture for 24 h, each half calvaria was
transferred to fresh medium with or without indicated reagents, and
cultured for an additional 72 h. For long bone culture, radii and
ulna were cultured in the same condition. The bone-resorbing activity
was determined by measuring the concentration of calcium in the
conditioned medium using a calcium kit (Calcium C test; Wako Pure
Chemicals, Osaka, Japan). The activity was expressed as an increase in
medium calcium (15). To detect osteoclasts, calvaria were fixed with
10% formalin and stained for tartrate-resistant acid phosphatase
(TRAP). The bone-resorbing activity expressed as an increase in medium
calcium changed in parallel with the number of TRAP-positive
osteoclasts in cultured calvaria (5, 15).
Northern Blot Analysis--
Total RNA was extracted from mouse
calvariae using the acid guanidium-phenol-chloroform method (15). For
Northern blotting, 20 µg of total RNA was resolved by electrophoresis
on a 1% agarose-formaldehyde gel and transferred onto a nylon
membrane, which was then hybridized with a 32P-labeled
cDNA probe as reported (4, 15). A 485-base pair fragment of mouse
MMP-13 cDNA and a 250-base pair fragment of human MMP-2 cDNA
were used as probes (15).
Gelatin Zymography--
Gelatinase activity in the conditioned
medium of calvarial cultures was analyzed by zymography as reported
previously (15). Aliquots (10 µl) were mixed with 5 µl of
non-reducing SDS-PAGE sample buffer, then subjected to SDS-PAGE using
10% polyacrylamide gel containing 0.6 mg/ml gelatin. After
electrophoresis, gels were incubated for 1 h in washing buffer (50 mM Tris-HCl containing 5 mM CaCl2 1 mM ZnCl2, and 2.5% Triton X-100) to remove
SDS, and then in the same buffer without Triton X-100 for 3 h.
Gels were then stained with Coomassie Brilliant Blue to detect enzyme
activity as a clear zone in a dark stained background.
Western Blot Analysis--
An aliquot of the conditioned medium
of calvarial cultures was subjected to SDS-PAGE using 10%
polyacrylamide gels, and separated proteins were transferred to a
polyvinylidene difluoride membrane (Hybond-PNDF, Amersham Pharmacia
Biotech). The membrane was first incubated for 18 h with 5% skim
milk in phosphate-buffered saline containing 0.1% Tween 20 at 4° C
to block nonspecific binding, and then incubated for 2 h with
polyclonal rabbit anti-MMP-13 antibody (kindly donated by Dr. Gillian
Murphy). After incubation with horseradish peroxidase-conjugated donkey
anti-rabbit Ig G for 1 h, immunoreactive bands were stained by an
ECL system (Amersham Pharmacia Biotech).
Assay of the Collagenase and Gelatinase Activities--
To
measure the collagenase and gelatinase activities, conditioned media of
calvarial cultures were treated for 4 h with 4-aminophenylmercuric acetate, which activates pro-MMPs to their respective active forms. The
collagenase and gelatinase activities were determined by measuring the
degradation of fluorescent isothiocyanate (FITC)-labeled type I and
type IV collagen using a type I or type IV collagenase assay kit (Yagai
Co.). One unit of these activities degrades 1 µg of each collagen/min
at 37° C.
Statistical Analysis--
Statistical analysis was carried out
by Student's t test, and the data are expressed as
means ± S.E.
PGE2 markedly stimulates osteoclast-mediated bone
resorption in vitro by enhancing both the formation and
function of osteoclasts. We previously showed in mouse bone marrow
cultures that PGE2 promoted osteoclast formation by a
cyclic AMP (cAMP)-mediated mechanism (2, 5). In fact, PGE2
acts on osteoblasts to elicit cAMP production. We also found previously
that IL-1 markedly induced the expression of MMPs including MMP-13,
MMP-2, and MMP-3 (stromelysin), and that this induction was associated
with an increase in bone-resorbing activity in mouse calvarial cultures
(15). In this study, we first used cultures of calvaria isolated from
normal ddy mice and examined the involvement of protein kinase A and
MMP induction in PGE2-induced bone resorption. Consistent
with the previous findings (2, 5), both PGE2 and
Bt2cAMP stimulated bone resorption in a
concentration-dependent manner in mouse calvarial cultures (Fig. 1A). Addition of an
inhibitor of protein kinase A, H89, markedly suppressed
PGE2-induced bone resorption (Fig. 1B),
indicating that a cAMP-dependent mechanism is essential for
bone resorption by PGE2 in the organ culture system. The
bone-resorbing activity in the control culture without PGE2
was not suppressed by H89 at all (Fig. 1B). To examine the
involvement of the induction of MMPs in PGE2-induced bone
resorption in this culture system, we subjected cultured calvaria and
its conditioned medium to Northern blot analysis, Western blot
analysis, and gelatin zymography of MMPs. Both PGE2 and
Bt2cAMP markedly increased expression of both MMP-13 and
MMP-2 mRNA on day 2 in mouse calvarial cultures (Fig. 1,
C and D). Consistently, Western blot analysis
showed the accumulation of MMP-13 protein in the medium of calvariae
treated with PGE2 or Bt2cAMP (Fig.
1C), and gelatin zymography revealed that MMP-2 activity
that was detected only marginally in the control culture was greatly
enhanced by treatment with either PGE2 or
Bt2cAMP (Fig. 1D). The potency of
PGE2 and Bt2cAMP in induction of MMPs was very
similar to that of IL-1 (15). To confirm that the MMPs expressed in
mouse calvariae had functional enzymatic activities, the collagenase
and gelatinase activities in the conditioned media were determined by
measuring the degradation of FITC-labeled type I and type IV collagen.
PGE2 and Bt2cAMP markedly increased both collagenase and gelatinase activities (Fig. 1, C and
D). To evaluate the role of MMP induction in
PGE2-induced bone resorption, we added BB94, an inhibitor
of MMPs, to mouse calvarial cultures treated with PGE2, and
examined its effects. As shown in Fig. 1B, BB94 markedly
suppressed bone-resorbing activity induced by PGE2, but the
activity of the control culture without PGE2 was not
altered by the inhibitor. These results indicate that the expression of
MMPs is essential for bone resorption, likely by promoting the
degradation of bone matrix, and that monitoring the induction of MMPs
is a useful measure for the bone-resorbing activity in mouse calvarial
cultures.
PGE2 thus causes bone resorption of cultured calvaria, and
protein kinase A and MMPs induction are involved in the process. However, the PGE receptor subtype(s) mediating this action remains unknown. To identify the responsible receptor subtype(s), we isolated calvariae from mice deficient individually in EP1, EP2, EP3, and EP4
receptor, and subjected them to bone resorption to PGE2. In calvariae from EP1(
Impaired Bone Resorption to Prostaglandin E2 in
Prostaglandin E Receptor EP4-knockout Mice*
§,§,,
,, and**
Department of Biochemistry, School of
Dentistry, Showa University, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo
142-8555, the ¶ Department of Physiological Chemistry, Faculty of
Pharmaceutical Science, Kyoto University, Kyoto 606-8501, the
Department of Pharmacology, Faculty of Medicine, Kyoto
University, Kyoto 606-8501, and the § Department of
Biochemistry, School of Pharmacy, Tokyo University of Pharmacy and Life
Science, Tokyo 192-0392, Japan
ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
/) neonates die within 72 h after birth due to
patent ductus arteriosus, suggesting that the EP4 receptor plays a role
in the regulation of the patency of this vessel (11, 14). On the other
hand, EP3 (/) mice failed to show a febrile response to various
pyrogens, suggesting that PGE2 mediates fever generation by
acting on the EP3 receptor (12).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Effects of PGE2 and
Bt2cAMP on bone resorption (A and
B) and induction of MMPs in mouse calvarial cultures
(C and D). A,
calvaria collected from 1-day-old ddy mice were cultured for 72 h
with various concentrations of PGE2 ( 
) or
Bt2cAMP (
). Conditioned media were collected, and
calcium contents were measured. Bone-resorbing activity was expressed
as the increase in the medium calcium. B, mouse calvariae
were cultured for 72 h with PGE2 (0.1-10
µM) in the presence or absence of 10 µM H89
(
), an inhibitor of protein kinase A, and 10 µM BB94
(
), an inhibitor of MMPs (, PGE2 only).
Bone-resorbing activity was calculated by measuring medium calcium
concentration. C, expression of MMP-13 mRNA and its
protein in mouse calvarial cultures. Mouse calvariae were cultured for
48 h with 1 µM PGE2 or 1000 µM Bt2cAMP. After culture, total RNA was
extracted from calvaria and Northern blotting was performed using
32P-labeled cDNA probes for MMP-13 (upper
panel). Protein lysates were extracted from calvaria and
Western blotting was performed using anti-MMP-13 antibody
(middle panel). Conditioned media were collected,
and collagenase activity was measured by the degradation of
FITC-labeled type I collagen after pre-treatment with 10 mM
4-aminophenylmercuric acetate to activate pro-MMPs, as described under
"Materials and Methods" (bottom panel).
D, expression of MMP-2 and gelatinase activity in mouse
calvarial cultures. After cultures were performed under the same
conditions as in C, the expression of MMP-2 mRNA was
examined by Northern blotting (upper panel), and
MMP-2 in the conditioned media was detected by gelatin zymography
(middle panel) as described under "Materials
and Methods." Gelatinase activity corresponding to pro-MMP-2 and
active-MMP-2 is indicated by arrows. Conditioned media were
also used to detect gelatinase activity measured by the degradation of
FITC-labeled type IV collagen (bottom panel).
Data are expressed as the means ± S.E. of four to nine
independent experiments.
/), EP2(/), and EP3(/) mice,
PGE2 stimulated bone resorption as in wild-type mice. In
contrast, a marked reduction in bone resorption was found in calvarial
culture from EP4 (/) mice (Fig.
2A). The
dose-dependent induction of bone-resorbing activity by
0.1-10 µM PGE2 was greatly diminished in
EP4(/) mice (Fig. 2B). PGE2 induced bone
resorption in calvaria from heterozygote EP4 (+/) mice to the same
level as that found in the bone from wild-type mice (data not shown).
To exclude a possibility of other defect(s) in the signal transduction
pathway, we examined the bone-resorbing activity of Bt2cAMP
in calvarial cultures from EP4 (/) and wild-type mice. As shown in
Fig. 2C, Bt2cAMP stimulated bone resorption
similarly in wild-type and EP4 (/) mice. Thus, the downstream
pathway from cAMP to the bone resorption appeared intact in EP4 (/)
mice, and the reduction of PGE2-induced bone resorption in
these mice is likely due to a lack of EP4 receptor (Fig. 2,
B and C). Calvaria from EP4(/) and wild-type
mice were cultured with PGE2 or Bt2cAMP, and
stained for TRAP to detect osteoclasts. In PGE2- and
Bt2cAMP-treated calvaria, numerous TRAP-positive osteoclasts were detected in the wild-type mice (Fig. 2D).
In contrast, in EP4(/) mice, osteoclasts were formed in
Bt2cAMP-treated calvaria, but not in
PGE2-treated calvaria (Fig. 2D). These
histological findings are consistent with the bone-resorbing activities
induced by PGE2 and Bt2cAMP shown in Fig. 2
(B and C).
View larger version (40K):
[in a new window]
Fig. 2.
Effects of PGE2 and
Bt2cAMP on bone resorption of calvaria from PGE receptor
(EP) knockout mice. A, mouse calvariae
were collected from 1-day-old wild-type mice and from EP1 ( /), EP2
(/), EP3 (/), and EP4 (/) mice, and cultured for 72 h
with or without 10 µM PGE2. Bone-resorbing
activity was expressed as the increase in medium calcium as described
under "Materials and Methods." Data are expressed as the means ± S.E. of 6-10 independent experiments. Results in EP4 (/) mice
were significantly different from the cultures treated with
PGE2 in wild-type mice (*, p < 0.001).
B, mouse calvaria were collected from wild-type mice ()
and EP4 (/) mice(
), and cultured for 72 h with 0.01-10
µM PGE2. Bone-resorbing activity was
measured. Data are expressed as the means ± S.E. of six cultures.
C, mouse calvariae were collected from wild-type and EP4
(/) mice, and bone-resorbing activity induced by 1000 µM Bt2cAMP was measured. Data are expressed
as means ± S.E. of six cultures. D, mouse calvariae
collected from wild-type and EP4 (/) mice were cultured for 72 h with 10 µM PGE2 or 1000 µM
Bt2cAMP. After culture, calvariae were fixed and stained
for TRAP to detect osteoclasts as described under "Materials and
Methods."
Induction of bone resorption by PGE2 was also examined in
long bone cultures using EP4(/) and wild-type mice.
PGE2 at 0.1-10 µM
dose-dependently stimulated bone resorption in long bone
cultures as well. In contrast, PGE2-induced bone resorption
was greatly impaired in long bone cultures from EP4(/) mice (Fig.
3A). Bt2cAMP, however, greatly stimulated bone resorption both in wild-type and
EP4(/) mice (Fig. 3B). These results are consistent with the data obtained by calvarial cultures shown in Fig. 2, confirming the
requisite role of EP4 for PGE2-induced bone resorption.
|
To further analyze the reduced bone resorption by PGE2 in
EP4 (/) mice, we examined the induction of MMPs by PGE2
by Western blot analysis and gelatin zymography of the culture media of
calvaria from these mice. Induction of MMP-2 and MMP-13 by
PGE2 was greatly diminished in EP4(/) mice compared
with the wild-type mice (Fig. 4). In
contrast, Bt2cAMP similarly induced MMP-2 and MMP-13 in both EP4(/) and wild-type mice (Fig. 4). This indicates that the
induction of MMPs is involved in PGE2-induced bone
resorption mediated by EP4.
|
Bone resorption is mediated by several processes, including osteoclast
differentiation, fusion and activation, and MMP-dependent matrix degradation. Recently Everts et al. (17) reported
that osteoclastic bone resorption depends on the activity of both
cysteine proteinases such as cathepsin K, and MMPs in calvaria, whereas long bone resorption depends on only cysteine proteinases. This suggests that there is a difference of osteoclast function in each
skeletal site. In this study, bone resorption induced by PGE2 was diminished not only in calvarial cultures but also
in long bone cultures (Figs. 2 and 3). Further studies are needed to
define whether the involvement of MMPs is different between PGE2-induced long bone resorption and calvarial bone
resorption in wild-type and EP4(/) mice. Osteoclast formation
induced by PGE2 was diminished in EP4(/) mice both in
calvarial cultures (Fig. 2D) and in bone marrow cultures
(data not shown). Therefore, the process of osteoclast differentiation
stimulated by PGE2 may also be involved in the mechanism of
impaired bone resorption to PGE2. More recently, Sakuma
et al. (18) reported that osteoclast formation was
diminished in the coculture of osteoblastic cells from EP4(/) mice
and spleen cells from wild-type mice. These findings indicate that
PGE2 stimulates bone resorption by a
cAMP-dependent mechanism mediated by EP4, and that the
induction of MMPs and osteoclast formation are involved in bone
resorption induced by PGE2.
As reported previously, all EP1(/), EP2(/), EP3(/), and
EP4(/) mice are born at the predicted Mendelian frequency. EP1(/), EP2(/), and EP3(/) mice grow normally, and no
apparent defects or abnormality is detected in bone by the soft x-ray
analysis (data not shown). Most EP4(/) neonates die within 72 h after birth by patent ductus arteriosus (11), which has precluded an
examination of bone tissues in adult EP4(/) mice. Reverse transcriptase-polymerase chain reaction analysis indicated that osteoblast-like cells isolated from calvaria of wild-type newborn mice
expressed all EPs mRNA, and the order of the expression levels was
EP4 > EP1 > EP2 > EP3 (data not shown). Because EP2
and EP4 stimulate adenylate cyclase in several types of cells, and cAMP production by osteoblasts is thought essential for the induction of
bone resorption by PGE2 (5-8), EP2 and EP4 have been
considered as the most likely receptors to mediate bone-resorbing
activity of PGE2. In this study, we have found a marked
reduction of bone-resorbing activity by PGE2 only in the
bone from EP4(/) mice. These observations indicate that
PGE2 stimulates bone resorption mainly by a
cAMP-dependent mechanism involving EP4. It should be noted,
however, that the PGE2-induced bone-resorbing activity was
not completely abolished in EP4 (/) mice. Some activity induced by
PGE2 remained in EP4(/) mice. Thus, a possible
involvement of other EPs in PGE2-induced bone resorption
cannot be excluded at present.
PGE2 is known to be a critical factor in bone formation and
resorption in vivo and in vitro (2, 3, 5, 19).
Recent studies suggest that PGE2 is involved in the
pathogenesis of certain metabolic bone diseases including osteoporosis
(20, 21). Cytokines such as IL-1 and IL-6 have bone-resorbing
activities and are likely involved in the pathogenesis of osteoporosis
(20, 22-25). Their bone-resorbing actions are at least partly
dependent on PGE2 production induced by these cytokines in
osteoblasts. One way to control PGE2-dependent
bone resorption may be therefore to regulate PGE2 production by osteoblasts. It is known that PGE2 synthesis
is regulated by the cytosolic phospholipase
A2-dependent release of arachidonic acid and
the COX-2-catalyzed conversion of arachidonic acid into
PGH2 (4, 26-28). COX-2 inhibitors have been therefore regarded potential candidates for the treatment of
PGE2-dependent bone resorption. This study
suggests an alternative possibility that specific antagonists for EP4
may be useful in regulating PGE-mediated metabolic bone diseases. This
possibility is currently being explored in our laboratories.
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ACKNOWLEDGEMENTS |
|---|
We are grateful to Dr. T. Maruyama (Ono Pharmaceutical Co. Ltd) for helpful discussion and to Dr. G. Murphy for the kind donation of anti-MMP-13 antibody.
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FOOTNOTES |
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* This work was supported by Grants-in-aid 08407060 (to T. S.) and 08457493 (to C. M.) from the Ministry of Science, Education and Culture of Japan.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. Section 1734 solely to indicate this fact.
** To whom all correspondence and reprint requests should be addressed. Tel.: 813-3784-8162; Fax: 813-3784-5555.
Published, JBC Papers in Press, April 3, 2000, DOI 10.1074/jbc.M002079200
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ABBREVIATIONS |
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The abbreviations used are: PG, prostaglandin; Bt2cAMP, dibutyryl cAMP; MMP, matrix metalloproteinase; PAGE, polyacrylamide gel electrophoresis; IL, interleukin; TRAP, tartrate-resistant acid phosphatase; FITC, fluorescein isothiocyanate; COX, cyclooxygenase.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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