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Department of Biochemistry, School of Dentistry, Showa University (C.M., T.T., T.S.), 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142; and Fuji Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd. (H.T., T.T., Y.O., Y.K., N.K.), Shizuoka 412, Japan; and the Division of Endocrinology and Metabolism, Department of Medicine, University of Connecticut Health Center (C.C.P., H.K., L.G.R.), Farmington, Connecticut 06030
Address all correspondence and requests for reprints to: Dr. Tatsuo Suda, Department of Biochemistry, School of Dentistry, Showa University, 1–5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan.
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and
IL-6 with sIL-6R caused marked induction of osteoclast formation and
PGE2 synthesis. These effects were suppressed by adding
neutralizing antibodies against IL-1 or IL-6R and were totally
abolished by adding nonsteroidal antiinflammatory drugs, such as
indomethacin and a selective cyclooxygenase-2 (COX-2) inhibitor
(NS398). In mouse osteoblastic cells, both IL-1 and IL-6 with sIL-6R
markedly induced messenger RNA expression of COX-2, but not COX-1, as
determined by Northern blot analysis, and luciferase activity in cells
stably transfected with a COX-2 promoter-luciferase fusion construct.
IL-6 and sIL-6R, when added separately, did not stimulate COX-2
messenger RNA expression. Simultaneous addition of IL-1 and IL-6
with sIL-6R to osteoblast cultures cooperatively induced transcription
of COX-2, which was associated with a marked increase in COX activity
measured by the conversion of arachidonic acid into PGE2.
The increased PGE2 synthesis by osteoblasts may play an
important role in osteoclastogenesis induced by submaximal doses of
IL-1 and IL-6. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF), and
PGE2 (1, 2, 3). IL-1 markedly stimulates osteoclastic bone
resorption in vitro and in vivo by enhancing both
osteoclast formation and function (1, 2, 3, 4). It is known that
PGE2 production by osteoblasts is involved in the
mechanism of osteoclast differentiation induced by IL-1 (3, 4). Other
cytokines, including IL-6, IL-11, and leukemia inhibitory factor, all
of which transduce their signals through the signal-transducing gp130
chain, also induce osteoclast formation in vitro (5, 6, 7).
IL-6 appears to act on osteoblastic cells, but not osteoclast
progenitors, to induce osteoclast differentiation (7). We reported that
the presence of soluble IL-6 receptor (sIL-6R) was essential for
osteoclast formation by IL-6 in cocultures of mouse bone marrow cells
and osteoblastic cells (5, 7) due to the lack of membrane-bound IL-6
receptors in osteoblasts under physiological conditions. In the course
of examining the mechanism of osteoclast differentiation by IL-6 and
IL-1, we found that submaximal doses of the two cytokines, neither of
which alone had much of an effect at that dosage, greatly stimulated
osteoclast formation in cocultures of bone marrow cells and
osteoblastic cells. This suggests that there is an interaction between
IL-1 and IL-6 in inducing osteoclast differentiation. However, no
common mechanism of IL-1 and IL-6 action in inducing osteoclast
differentiation has been reported. PG synthesis is regulated by two successive metabolic steps; the release of arachidonic acid from membranous phospholipids and its conversion to prostanoids. Phospholipase A2 is the enzyme responsible for arachidonic acid release, and cyclooxygenase (COX) is a rate-limiting enzyme for the conversion of arachidonic acid to prostanoids (8, 9). Two COX genes, COX-1 and COX-2, have been identified (10, 11). COX-1 is constitutively expressed in many mammalian tissues (10). In contrast, COX-2 is generally undetectable under physiological conditions, but is markedly induced by several cytokines and growth factors (8, 9, 10, 11). Both COX-1 and COX-2 are expressed in osteoblastic cells. We reported that IL-1 stimulates messenger RNA (mRNA) expression of COX-2, but not COX-1, in osteoblastic cells, and that COX-2 is the major enzyme regulating PG synthesis in response to several bone-resorbing factors, such as IL-1, basic fibroblast growth factor, and PGE2 (12, 13, 14, 15). Therefore, COX-2-dependent PG synthesis by osteoblasts is considered to play a key role in bone resorption associated with inflammation.
Estrogen deficiency causes a marked bone loss by stimulating osteoclastic bone resorption. Recent studies have focused on the involvement of IL-1 and IL-6 in osteoclastic bone resorption due to estrogen deficiency. The administration of IL-1 receptor antagonist to ovariectomized (OVX) rats decreased bone loss (16, 17). The increased number of osteoclasts in OVX mice was normalized by administration of a neutralizing antibody against IL-6 (18, 19). We reported that the bone-resorbing activity present in bone marrow supernatants from OVX mice was much higher than that in bone marrow supernatants from sham mice (20). In mouse calvarial cultures, bone marrow supernatants from OVX mice had a greater effect on COX-2 mRNA expression and PGE2 synthesis than those from sham mice (21). These results suggest that PGE2 is also involved in bone resorption due to estrogen deficiency.
In this study, we examined the possible involvement of PGE2 synthesis by osteoblastic cells in IL-6-induced osteoclast formation. Both IL-1 and IL-6 in the presence of sIL-6R cooperatively stimulated osteoclast formation and PGE2 production in cocultures of mouse bone marrow cells and osteoblastic cells. In osteoblasts, not only IL-1, but also IL-6 in the presence of sIL-6R, markedly stimulated COX-2 gene transcription. IL-1 and IL-6 with sIL-6R cooperatively induced COX-2 expression, which resulted in marked stimulation of COX activity. The COX-2-dependent PG synthesis involved in osteoclastogenesis may be additively induced by IL-1 and IL-6.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and neutralizing antibody
against human IL-1 were purchased from Genzyme (Cambridge, MA).
Recombinant mouse IL-6 and recombinant mouse sIL-6R were prepared from
Chinese hamster ovary cells transfected with a mouse IL-6 complementary
DNA (cDNA) expression vector and a mouse sIL-6R cDNA expression vector,
respectively, as previously reported (5). Recombinant human IL-6 and
human sIL-6R were purchased from R&D Systems (Minneapolis, MN).
Neutralizing antibody against mouse IL-6R (MR16–1) was prepared as
described previously (22). Arachidonic acid was purchased from Sigma
Chemical Co. (St. Louis, MO). NS398 was purchased from Calbiochem (La
Jolla, CA). All other chemicals were of analytical grade.
Culture of primary mouse osteoblastic cells
Primary osteoblastic cells were isolated from 1-day-old mouse
calvaria after five routine sequential digestions with 0.1%
collagenase (Wako Pure Chemicals, Osaka, Japan) and 0.2% dispase (Godo
Shusei, Tokyo, Japan) as previously described (12). Osteoblasts
isolated from fractions 3–5 were combined and cultured in MEM
supplemented with 10% FBS at 37 C in a humidified atmosphere of 5%
CO2 in air.
Coculture of mouse bone marrow cells and osteoblastic cells
Primary osteoblastic cells (1 x 104) were
cocultured with bone marrow cells (2 x 105) for 7
days in the well of 48-well culture plates with 0.3 ml MEM
containing 10% FBS as previously reported (3, 5). On day 4, medium was
changed to the respective fresh medium containing each test chemical.
On day 7, adherent cells were fixed and stained for tartrate-resistant
acid phosphatase (TRAP), and the number of TRAP-positive
osteoclast-like multinucleated cells formed was counted as previously
described (3, 5). The conditioned media collected on days 4 and 7 were
combined and used for the determination of PGE2
levels.
Measurement of PGE2 content
The concentration of PGE2 in the culture
medium was determined using a RIA kit (NEK-020, DuPont-New England
Nuclear, Boston, MA). The detection limit of the
PGE2 RIA kit was 0.5 ng/ml.
Northern blot analysis
Primary osteoblastic cells were cultured for 24 h in MEM
supplemented with 0.1% FBS, then incubated for 3 h with or
without cytokines. A DNA construct containing 371 bp of the murine
COX-2 promoter and 70 bp of downstream untranslated DNA fused to a
luciferase reporter gene (23) was kindly provided by Dr. H. Herschman
(University of California, Los Angeles, CA). A mouse osteoblastic cell
line, MC3T3-E1, stably transfected with this construct, as described
below, was cultured for 24 h in serum-free DMEM, then incubated
for 30–180 min with or without cytokines. Total cellular RNA was
extracted using the acid guanidium-phenol-chloroform method (12). For
Northern blotting, 10 µg total RNA were resolved by electrophoresis
in a 1% agarose-formaldehyde gel and transferred onto nylon membranes
(Hybond N, Amersham, Arlington Heights, IL), then hybridized with a
32P-labeled cDNA probe as previously reported (12). The
signals were densitometrically quantified using a Bioimage analyzer
(BAS-2000, Fuji Film, Tokyo, Japan). Mouse COX-1 and COX-2 cDNA probes
were purchased from Oxford Biomedical Research (Oxford, MI). Mouse
COX-2 cDNA was provided by Dr. H. Herschman (University of California,
Los Angeles), and mouse COX-1 cDNA was provided by Dr. W. Smith
(Michigan State University, East Lansing, MI). Mouse IL-6 and mouse
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probes were
amplified by PCR using the respective amplimer sets from Clontech (Palo
Alto, CA). Luciferase cDNA was amplified by PCR (sense primer,
5'-CGCTGGAGAGCAACGCATAAGGTTATG-3'; antisense primer,
5'-TAGTCTCAGTGAGCCCATATCCTTGTCG-3').
Assay for COX activity
Primary osteoblastic cells and MC3T3-E1 cells were cultured for
15 h on 48-well culture plates containing MEM supplemented with
0.1% FBS, then treated for 5 h with or without cytokines in
MEM containing 0.1% FBS. At the end of culture, cells were washed
twice with MEM and incubated for 15 min with 10 µM
arachidonic acid in MEM containing 0.1% FBS. Conditioned media were
collected for the determination of PGE2
levels.
Determination of COX-2 promoter activity
The COX-2 promoter-luciferase fusion construct containing 371 bp
of the 5'-flanking sequence and 70 bp of downstream untranslated DNA
(P2-Luc371) (23) was purified by CsCl banding and cotransfected with
pSV2-neo into cultured MC3T3-E1 cells using Lipofectamine (Life
Technologies, Grand Island, NY). After selection using G418 for 2
weeks, stable colonies were pooled and used for luciferase assay as
previously reported (13). Cells (5 x 103) were plated
in six-well dishes and grown for 6 days in DMEM containing 10% FBS.
They were precultured for 24 h in serum-free DMEM with 1 mg/ml
BSA, then treated with human IL-1 and/or human IL-6 with sIL-6R.
Luciferase activity was measured in soluble cell extracts prepared with
a luciferase detection kit (Promega, Madison, WI) using an automatic
injection luminometer (Berthold Lumat, Wallac, Gaithersburg, MD).
Activity was normalized to total protein measured with a BCA protein
assay kit (Pierce Chemical Co., Rockford, IL).
Statistical analysis
Statistical analysis was carried out using ANOVA, and the
significance of differences between two groups was determined by a
post-hoc test using the Bonferroni/Dunn method.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, at 30–1000 pg/ml,
also induces osteoclast formation in this coculture system (3). When
submaximal doses of IL-1 (30 pg/ml) or IL-6 (20 ng/ml) together with
sIL-6R (250 ng/ml) were separately added to the coculture, osteoclast
formation was detected, but only slightly (Fig. 1
).
Simultaneous addition of these cytokines caused a marked cooperative
effect on osteoclast formation, and the potency was equivalent to the
maximal effects induced by either 1000 pg/ml IL-1 or 200 ng/ml IL-6
with sIL-6R (250 ng/ml; Fig. 1). Furthermore, treatment with a
submaximal dose (30 pg/ml) of IL-1 and a maximal dose (200 ng/ml) of
IL-6 with sIL-6R (250 ng/ml) had an even greater effect on osteoclast
formation (Fig. 1).
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shows the effects of neutralizing antibodies
against human IL-1 and mouse IL-6R on osteoclast formation induced
by submaximal doses of IL-1 (30 pg/ml) and IL-6 (20 ng/ml) in the
presence of sIL-6R (250 ng/ml). These neutralizing antibodies have been
proven to abolish specifically the biological function of IL-1 and
IL-6, respectively (22). Both antibodies suppressed the osteoclast
formation induced by submaximal doses of IL-1 and IL-6 in the
presence of sIL-6R when they were added separately (Fig. 2).
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shows the effects on PGE2 production of IL-1
and IL-6 with sIL-6R added separately or in combination to the
coculture. PGE2 production was examined by
measuring the level of PGE2 in conditioned
medium, as shown in Fig. 1
. Not only IL-1, but also IL-6, in the
presence of sIL-6R markedly stimulated PGE2
production in the cocultures (Fig. 3). When submaximal doses of IL-1
(30 pg/ml) and IL-6 (20 ng/ml) together with sIL-6R (250 ng/ml) were
added simultaneously, PGE2 production was
increased to a level equivalent to that induced by 1000 pg/ml IL-1
(Fig. 3).
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(30 pg/ml)
and IL-6 (20 ng/ml) in the presence of sIL-6R (250 ng/ml; Fig. 4). Under these conditions, the level of
PGE2 produced by low doses of IL-1 and IL-6 was
35 ng/ml, and this was completely suppressed by adding indomethacin
(data not shown). When the same amount (35 ng/ml) of
PGE2 was added to the coculture with indomethacin
or NS-398, osteoclast formation was completely restored (Fig. 4). These
results indicate that the endogenous production of
PGE2 is a prerequisite for the osteoclast
formation induced by submaximal doses of IL-1 and IL-6 with
sIL-6R.
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MEM
containing 0.1% FBS for 24 h, then treated for 3 h with or
without IL-6 and sIL-6R. IL-6 (200 ng/ml) in the presence of sIL-6R
(250 ng/ml) significantly induced COX-2 mRNA expression (Fig. 5). When submarginal doses of IL-1 (30 pg/ml) and
IL-6 (20 ng/ml) with sIL-6R (250 ng/ml) were separately added to the
coculture, there was slight expression of COX-2 mRNA. sIL-6R (250
ng/ml) alone had no effect. Simultaneous addition of the submaximal
doses of IL-1 and IL-6 together with sIL-6R caused a marked increase
in COX-2 mRNA expression in osteoblasts. The potency was greater than
that induced by 1000 pg/ml IL-1 (Fig. 5).
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and/or IL-6 in
the presence of sIL-6R in MEM containing 0.1% FBS, they were washed
and incubated for 15 min in MEM containing 10
µM arachidonic acid. The COX activity, as
measured by PGE2 production, was induced not only
by IL-1 but also by IL-6 in the presence of sIL-6R (Table 1). Consistent with the data shown in Fig. 5, simultaneous treatment with submaximal doses of IL-1 (30 pg/ml) and
IL-6 (20 ng/ml) in the presence of sIL-6R (250 ng/ml) induced even
greater COX activity than that produced by a high dose of IL-1 (Table 1). This indicates that the COX-2 enzyme induced by submaximal doses of
IL-1 and IL-6 together with sIL-6R is capable of converting arachidonic
acid into PGE2 in osteoblastic cells. The
induction of COX activity by submaximal doses of IL-1 and IL-6 was
similar to that by a high dose of IL-1 in MC3T3-E1 cells (Table 1).
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(10 ng/ml; T/C ratios
for luciferase activity were 2.8 and 3.1, respectively). Neither IL-6
nor sIL-6R stimulated luciferase activity, when they were added
separately. Simultaneous treatment with IL-1 (10 ng/ml) and IL-6
(200 ng/ml) in the presence of sIL-6R (250 ng/ml) enhanced luciferase
activity in the cells transfected with P2-Luc371 (T/C ratio was 6.2;
Fig. 6).
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). IL-1 also stimulated the expression of luciferase
and COX-2 mRNAs at 30–180 min. Simultaneous treatment with IL-1 and
IL-6 in the presence of sIL-6R cooperatively induced the expression of
luciferase and COX-2 mRNAs in MC3T3-E1 cells transfected with P2-Luc371
(Fig. 7). Expression of COX-1 mRNA could be detected in the cells, but
it was not affected appreciably by treatment with IL-1 and/or IL-6
(Fig. 7). In addition, we found that IL-6 with sIL-6R stimulated IL-6
mRNA expression at 30–60 min (Fig. 7). As reported previously, IL-1
induced IL-6 mRNA expression at 120–180 min. The expression of IL-6
mRNA was synergistically enhanced by simultaneous addition of IL-1
and IL-6 in the presence of sIL-6R at 30–60 min, suggesting that
autoamplification of the IL-6 gene by IL-6 is involved in the mechanism
of additive production of PGE2 by IL-1 and
IL-6.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-6 in the
presence of sIL-6R cooperatively induced osteoclast formation in the
coculture as well as PGE2 production by
osteoblasts. Osteoclast formation induced by IL-1 and/or IL-6 appeared
to depend on their ability to induce COX-2 gene transcription in
osteoblasts.
We reported that IL-6 alone could not induce osteoclast formation in
cocultures of mouse bone marrow cells and osteoblastic cells, but
sIL-6R strikingly triggered osteoclast formation by IL-6 (5). We also
reported that mouse primary osteoblastic cells expressed a very low
level of membrane-bound IL-6 receptors, which was not enough for
IL-6-mediated signal transduction (7). In this study, IL-6 alone
stimulated neither COX-2 mRNA expression nor COX-2 promoter activity in
osteoblastic cells, but sIL-6R triggered COX-2 mRNA expression by IL-6.
This is consistent with the previous findings on osteoclast formation
and signal transduction of gp130-related cytokines in osteoblasts (27).
Romas et al. (28) reported that IL-1, TNF,
PGE2, PTH, and 1,25-dihydroxyvitamin
D3 similarly induced IL-11 production by osteoblasts, and
that neutralizing antibody against mouse gp130 inhibited osteoclast
formation induced by these factors completely or partially. Girasole
et al. (6) also reported that PTH and
1,25-dihydroxyvitamin D3 stimulated IL-11 production in
bone marrow stromal cell cultures, and antibody against IL-11
suppressed osteoclast formation induced by these factors. In the
present study, osteoclast formation induced by submaximal doses of IL-1
and IL-6 in the presence of sIL-6R was completely suppressed by
indomethacin and a selective COX-2 inhibitor (NS-398). Osteoclast
formation induced by IL-11 was also inhibited by indomethacin (data not
shown), indicating that PGE2 production is
essential for IL-11-induced osteoclast formation. Pilbeam et
al. (14) reported autoamplification of COX-2 in osteoblastic cells
by PGE2. This suggests that
PGE2 produced by osteoblasts in response to IL-1
and IL-6 stimulates COX-2 expression in osteoblasts, which, in turn,
causes a marked increase in PGE2 synthesis.
PGE2 production by osteoblasts can be regulated by many factors, including IL-1, PTH, basic fibroblast growth factor, TGFs, and PGE per se (12, 13, 14, 15, 29, 30). COX-2 expression appears responsible for bone resorption induced by some of these factors (12, 13). The present study suggests that COX-2 is preferentially responsible for the IL-1-induced PG synthesis by osteoblasts. However, the possibility that COX-1 is also involved in the IL-1-induced PG synthesis by osteoblasts cannot be ruled out completely at present, as constant expression of COX-1 mRNA was detected as well. The COX-1 protein was weakly, but constantly, expressed in osteoblasts, but it was not affected by treatment with IL-1 (31).
We found that IL-6 induces transcriptional activation of COX-2 in the
presence of sIL-6R in osteoblasts. The 5'-flanking region of the COX-2
gene promoter contains various putative transcriptional regulatory
elements such as cAMP responsive element, nuclear factor IL-6
(NF-IL-6), activator protein-2, specificity protein 1 (Sp1), and
nuclear factor-
B (NFB) (32, 33). Of these regulatory elements,
cAMP response element, and NF-IL-6 have been reported to act as
positive regulatory elements for COX-2 transcription (30, 32). In mouse
osteoblasts, Yamamoto et al. (34) reported that both NF-IL-6
and NFB were responsible for COX-2 transcription induced by TNF.
The 371-bp proximal region used in the present study contains an
NF-IL-6, but no NFB response element. This suggests that NFB is
not crucial for COX-2 transcription induced by IL-1 and IL-6.
IL-6-induced gp130 signals activate both the tyrosine kinase
JAK2-signal transducer and activator of transcription (STAT) cascade
and Ras-dependent MAP kinase cascade, and the latter cascade leads to
the activation of NF-IL-6 (35). Further studies are needed to identify
the elements regulated by IL-1 and IL-6 in the mouse COX-2 gene
promoter.
Bone loss caused by estrogen deficiency is thought to be due to the
increased bone resorption stimulated by cytokines such as IL-1 and
IL-6. Pacifici and his co-workers reported that the administration of
IL-1 receptor antagonist to OVX rats decreased bone resorption (17).
Manolagas and his co-workers reported that the increased bone
resorption in OVX mice was restored by giving mice anti-IL-6 antibody
in vivo (19). We reported that bone-resorbing activity
present in bone marrow supernatants from OVX mice was much higher than
that from sham mice (20) and was suppressed by indomethacin as well as
antibodies against IL-1 and IL-6. The bone marrow supernatants from OVX
mice had a greater effect than the supernatants from sham mice on
inducing COX-2 expression and PG synthesis in mouse calvarial cultures
(21). These results suggest that synergistic effects of IL-1 and IL-6
on PGE2 synthesis may be critical in the
mechanism of bone resorption in estrogen deficiency. It was also
reported that bone loss in rheumatoid arthritis patients was linked to
the increased levels of bone-resorbing cytokines, including IL-1, IL-6,
and TNF in synovial fluids (24, 25, 36). Expression of COX-2 mRNA
occurred in synovial cells of rheumatoid arthritis patients (37),
suggesting that PGs are involved in bone resorption in rheumatoid
arthritis patients as well.
In conclusion, IL-6 induces COX-2 transcription in the presence of sIL-6R in mouse osteoblasts, and this induction appears to be involved in IL-6-induced osteoclast formation. The additive stimulation of osteoclast differentiation by the submaximal doses of IL-1 and IL-6 may be explained by the greater induction of COX-2-dependent PG synthesis by a combination of these cytokines, although COX-1 may also play a role. How IL-1 and IL-6 interact to induce COX-2 transcription is interesting and is currently under investigation in our laboratories.
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Acknowledgments |
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Footnotes |
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Received December 23, 1996.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and transforming growth factor in osteoblastic
MC3T3–E1 cells. J Bone Miner Res 9:817–823[Medline]
B and nuclear
factor-interleukin-6 in the tumor necrosis factor -dependent
induction of cyclooxygenase-2 in MC3T3–E1 cells. J Biol Chem 270:31315–31320 (CA2) vs. placebo in
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C. Miyaura, M. Inada, T. Suzawa, Y. Sugimoto, F. Ushikubi, A. Ichikawa, S. Narumiya, and T. Suda Impaired Bone Resorption to Prostaglandin E2 in Prostaglandin E Receptor EP4-knockout Mice J. Biol. Chem., June 23, 2000; 275(26): 19819 - 19823. [Abstract] [Full Text] [PDF]
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 D. A. Bushinsky, W. R. Pa |
) and
COX-2 (
) mRNAs is shown as relative intensity compared to that of
the control culture.
