Endocrinology Vol. 140, No. 11 5120-5125
Copyright © 1999 by The Endocrine Society
Mitogen-Activated Protein (MAP) Kinases Are Involved in Interleukin-1 (IL-1)-Induced IL-6 Synthesis in Osteoblasts: Modulation Not of p38 MAP Kinase, But of p42/p44 MAP Kinase by IL-1-Activated Protein Kinase C1
Masaichi Miwa,
Osamu Kozawa,
Haruhiko Tokuda and
Toshihiko Uematsu
Department of Internal Medicine, Gifu Social Insurance Hospital
(M.M.), Kani, Gifu 509-0206; the Department of Pharmacology, Gifu
University School of Medicine (O.K., T.U.), Gifu 500-8705; and the
Department of Internal Medicine, Chubu National Hospital: National
Institute for Longevity Sciences (H.T.), Obu, Aichi 474-8511,
Japan
Address all correspondence and requests for reprints to: Dr. Osamu Kozawa, Department of Pharmacology, Gifu University School of Medicine, Gifu 500-8705, Japan.
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Abstract
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We previously reported that interleukin-1
(IL-1)-induced
activation of protein kinase C (PKC) via phosphatidylcholine-specific
phospholipase C (PC-PLC) limits IL-6 synthesis induced by IL-1
itself in osteoblast-like MC3T3-E1 cells. In the present study, we
further investigated the mechanism behind IL-1-induced IL-6
synthesis in MC3T3-E1 cells. IL-1 time-dependently stimulated the
phosphorylation of both p42/p44 mitogen-activated protein (MAP) kinase
and p38 MAP kinase. PD98059, a specific inhibitor of the upstream
kinase that activates p42/p44 MAP kinase, inhibited the IL-1-induced
IL-6 synthesis as well as the phosphorylation of p42/p44 MAP kinase
induced by IL-1. SB203580, a specific inhibitor of p38 MAP kinase,
also reduced both the phosphorylation of p38 MAP kinase and the IL-6
synthesis. 1-Oleoyl-2-acetylglycerol, an activator of PKC, suppressed
the IL-1-induced IL-6 synthesis. Calphostin C, a specific inhibitor
of PKC, or D-609, a specific inhibitor of PC-PLC, significantly
enhanced the IL-1-induced phosphorylation of p42/p44 MAP kinase
without affecting the phosphorylation of p38 MAP kinase. The
phosphorylation of p42/p44 MAP kinase by IL-1 was markedly increased
in PKC-down-regulated MC3T3-E1 cells. Neither
12-O-tetradecanoylphorbol-13-acetate, known to be an
activator of PKC, nor 1-oleoyl-2-acetylglycerol affected the
phosphorylation of p38 MAP kinase induced by IL-1. These results
strongly suggest that IL-1-induced IL-6 synthesis is mediated via
activations of both p42/p44 MAP kinase and p38 MAP kinase in
osteoblasts, and that PKC activated by IL-1 itself negatively
regulates IL-6 synthesis at a point upstream from p42/p44 MAP kinase.
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Introduction
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INTERLEUKIN-1 (IL-1) is known as one of
multifunctional cytokines responsible for inflammation, infection, and
cancer, and it induces numerous physiological effects on a wide variety
of cells (1, 2). In bone, it is well recognized that IL-1 is a potent
bone-resorptive agent (3, 4). Bone metabolism is maintained by two
types of functional bone cells, osteoblasts and osteoclasts, which are
responsible for bone formation and bone resorption, respectively (3).
Accumulating evidence indicates that osteoblasts, rather than
osteoclasts, possess receptors for many bone-resorptive agents (3). It
has been reported that IL-1 as well as other bone-resorptive agents,
such as PTH, tumor necrosis factor-, and platelet-derived growth
factor, stimulate the production of IL-6 in cultured osteoblasts
(4, 5, 6, 7). As IL-6 has been shown to stimulate bone resorption and induce
osteoclast formation (4, 8), it is currently recognized that IL-6
secreted from osteoblasts plays a key role as an important downstream
effector of bone-resorptive agents. We have recently reported that
IL-1 activates protein kinase C (PKC) through phosphatidylcholine
hydrolysis by phosphatidylcholine-specific phospholipase C (PC-PLC) in
osteoblast-like MC3T3-E1 cells, and that the PKC activation then limits
IL-6 synthesis induced by IL-1 itself (9). However, the stimulative
signaling pathway underlying the IL-1-induced IL-6 synthesis in
osteoblasts has not yet been clarified.
The mitogen-activated protein (MAP) kinase family plays important roles
in intracellular signaling of a variety of agonists (10). It is well
known that MAP kinases are activated on dual phosphorylation of
threonine and tyrosine (10). In osteoblastic cells, MAP kinases have
been reported to be involved in various intracellular signaling
pathways (11, 12, 13, 14, 15). As for intracellular signaling of IL-1, IL-1 has
been shown to activate p42/p44 MAP kinase in rabbit articular
chondrocytes (16). In addition, it has recently been reported that p38
MAP kinase is involved in IL-1ß-induced IL-6 gene expression in human
fibroblast-like synoviocytes (17). Furthermore, IL-1ß reportedly
activates extracellular signal-regulated kinase, the so-called p42/p44
MAP kinase, and c-Jun NH2-terminal kinase pathways in
normal human osteoblastic cells and rat osteosarcoma UMR-106 cells
(15). Thus, the present study was conducted to clarify whether p42/p44
MAP kinase and/or p38 MAP kinase are involved in the IL-1-induced
IL-6 synthesis in osteoblast-like MC3T3-E1 cells. We here show that
IL-1-induced IL-6 synthesis is mediated via activations of both
p42/p44 MAP kinase and p38 MAP kinase in these cells, and that the PKC
activation negatively regulates IL-6 synthesis at a point upstream from
p42/p44 MAP kinase.
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Materials and Methods
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Materials
IL-1 and potassium
tricyclo-(5,2,1,0)-decyl-[9(8)-xanthogenate] (D-609) were obtained
from Funakoshi Pharmaceutical Co. (Tokyo, Japan).
2'-Amino-3'-methoxyflanone (PD98059) and
4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole
(SB203580) were obtained from Calbiochem (La Jolla, CA).
Calphostin C and 12-O-tetradecanoylphorbol-13-acetate (TPA)
were purchased from Sigma Chemical Co. (St. Louis, MO).
1-Oleoyl-2-acetylglycerol (OAG) was purchased from Nacalai Tesque
(Kyoto, Japan). Phospho-specific p42/p44 MAP kinase antibodies (rabbit
polyclonal IgG, affinity purified), p42/p44 MAP kinase antibodies
(rabbit polyclonal IgG, affinity purified), phospho-specific p38 MAP
kinase antibodies (rabbit polyclonal IgG, affinity purified), and p38
MAP kinase antibodies (rabbit polyclonal IgG, affinity purified) were
purchased from New England Biolabs, Inc. (Beverly, MA).
The mouse IL-6 enzyme immunoassay kit, the enhanced chemiluminescence
Western blotting detection system, the PKC assay system, and
[
-32P]ATP (3000 Ci/mmol) were purchased from
Amersham Pharmacia Biotech Japan (Tokyo, Japan). Other
materials and chemicals were obtained from commercial sources. PD98059,
SB203580, calphostin C, and TPA were dissolved in dimethylsulfoxide.
The maximal concentration of dimethylsulfoxide was 0.1%, which did not
affect the assay for IL-6 or the analyses of MAP kinases.
Cell culture
Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse
calvaria (18, 19) were maintained as previously described (20).
Briefly, the cells were cultured in MEM containing 10% FCS at 37 C
in a humidified atmosphere of 5% CO2-95% air. The cells
(5 x 104) were seeded into 35-mm diameter dishes in 2
ml MEM containing 10% FCS. After 5 days, the medium was exchanged
for 2 ml MEM containing 0.3% FCS. The cells were used for
experiments after 48 h. When indicated, the cells were pretreated
with 0.1 µM TPA for 24 h, as previously reported
(21).
Assay for IL-6
The cultured cells were pretreated with various doses of
PD98059, SB203580, or OAG for 20 min, and then stimulated by 30 ng/ml
(
1.66 nM) IL-1 or vehicle in 1 ml MEM containing
0.3% FCS for the indicated periods. The conditioned medium was
collected, and IL-6 in the medium was measured by an IL-6 enzyme
immunoassay kit.
Analyses of MAP kinases
The cultured cells were stimulated by IL-1 in 1 ml MEM for
the indicated periods. The cells were washed twice with 1 ml PBS and
then lysed, homogenized, and sonicated in a lysis buffer containing
62.5 mM Tris/HCl (pH 6.8), 2% SDS, 50 mM
dithiothreitol, and 10% glycerol. The cytosolic fraction was collected
as a supernatant after centrifugation at 125,000 x g
for 10 min at 4 C. SDS-PAGE was performed by the method of Laemmli (22)
in 10% polyacrylamide gel. Western blotting analysis was performed as
described previously (23) using phospho-specific p42/p44 MAP kinase
antibodies, p42/p44 MAP kinase antibodies, phospho-specific p38 MAP
kinase antibodies or p38 MAP kinase antibodies, and peroxidase-labeled
antibodies raised in goat against rabbit IgG as second antibodies.
Peroxidase activity on the nitrocellulose sheet was visualized on x-ray
film using the enhanced chemiluminescence Western blotting detection
system. When indicated, the cells were pretreated with PD98059,
SB203580, calphostin C, D-609, TPA, or OAG for 20 min.
Assay for PKC activity
The cultured cells were pretreated with calphostin C for 20 min
and then stimulated by IL-1 at 37 C for 10 min in 1 ml assay buffer
[5 mM HEPES (pH 7.4), 150 mM NaCl, 5
mM KCl, 5.5 mM glucose, 0.8 mM
MgSO4, and 1 mM CaCl2] containing
0.01% BSA. The reaction was terminated by aspirating the medium, and
PKC activity was determined using a PKC assay system.
Statistical analysis
The data were analyzed by one-way ANOVA, followed by
Bonferroni’s method for multiple comparison between pairs.
P < 0.05 was considered significant. All data are
presented as the mean ± SEM of triplicate
determinations. Each experiment was repeated three times with similar
results.
Determinations
The absorbance of enzyme immunoassay samples was measured at 450
nm with an EL 340 Bio Kinetic Reader (Bio-Tek Instruments, Inc., Winooski, VT). The radioactivity of 32P
samples for PKC activity was determined with an LS-6500IC liquid
scintillation spectrometer (Beckman Coulter, Inc., Palo
Alto, CA).
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Results
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Effects of IL-1 on the phosphorylation of p42/p44 MAP kinase or
p38 MAP kinase in MC3T3-E1 cells
To investigate whether IL-1 activates p42/p44 MAP kinase and/or
p38 MAP kinase in osteoblast-like MC3T3-E1 cells, we examined the
effect of IL-1 on the phosphorylation of p42/p44 MAP kinase and p38
MAP kinase by Western blotting analysis. IL-1 time dependently
elicited the phosphorylation of p42/p44 MAP kinase and p38 MAP kinase
(Fig. 1
). The phosphorylation of both
p42/p44 MAP kinase and p38 MAP kinase were detectable for 3–10 min
after the stimulation of IL-1. The maximal effects of IL-1 on the
phosphorylation of p42/p44 MAP kinase and p38 MAP kinase were observed
at 10 and 5 min, respectively (Fig. 1). Despite using the antibodies
that recognize both p42 and p44 MAP kinases, the band of p44 MAP kinase
appeared much fainter than that of p42 MAP kinase in MC3T3-E1
cells.
Effect of PD98059 or SB203580 on IL-1-induced IL-6 synthesis in
MC3T3-E1 cells
We previously showed that IL-6 synthesis is stimulated by
IL-1 in osteoblast-like MC3T3-E1 cells (9). To clarify whether the
activation(s) of p42/p44 MAP kinase and/or p38 MAP kinase is involved
in IL-1-induced IL-6 synthesis in these cells, we examined the
effect of PD98059, a specific inhibitor of the upstream kinase that
activates p42/p44 MAP kinase (24, 25), or SB203580, an inhibitor of p38
MAP kinase (26, 27), on the IL-6 synthesis induced by IL-1. As
previously reported (9), IL-1 stimulated IL-6 synthesis in a
time-dependent manner up to 24 h. PD98059, which alone had little
effect on IL-6 synthesis, significantly inhibited IL-1-induced IL-6
synthesis (Fig. 2A). The inhibitory
effect of PD98059 on IL-1-induced IL-6 synthesis was dose dependent
in the range between 0.1–50 µM (Fig. 2B). The maximal
inhibitory effect of PD98059 was achieved at 20 µM, a
dose that led to about 75% reduction of the IL-1 effect, and this
effect was not statistically different from that of 50
µM. In addition, SB203580, which alone hardly affected
the basal level of IL-6 synthesis, markedly reduced IL-1-induced
IL-6 synthesis (Fig. 3A). The inhibitory
effect of SB203580 on IL-1-induced IL-6 synthesis was dose dependent
in the range between 0.1–30 µM (Fig. 3B). The maximal
inhibitory effect of SB203580 was observed at 30 µM, a
dose that led to an almost complete reduction of the IL-1 effect. We
confirmed that PD98059 reduced the IL-1-induced phosphorylation of
p42/p44 MAP kinase (Fig. 4A), and that
SB203580 suppressed the phosphorylation of p38 MAP kinase induced by
IL-1 in these cells (Fig. 4B).
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Figure 4. Effects of PD98059 or SB203850 on the
IL-1-induced phosphorylation of p42/p44 MAP kinase and p38 MAP
kinase in MC3T3-E1 cells. A, The cultured cells were pretreated with 20
µM PD98059 or vehicle for 20 min, and then stimulated by
30 ng/ml IL-1 or vehicle (lanes 1, 3, and 5) for 5 min. Upper
panel, Antiphospho-specific p42/p44 MAP kinase antibodies.
Lower panel, Anti-p42/p44 MAP kinase antibodies. B, The
cultured cells were pretreated with 3 µM SB203580 or
vehicle for 20 min, and then stimulated by 30 ng/ml IL-1 or for 5
min. Upper panel, Antiphospho-specific p38 MAP kinase
antibodies. Lower panel, Anti-p38 MAP kinase antibodies.
The results are representative of triplicate independent experiments.
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Effect of calphostin C or D-609 on IL-1-induced phosphorylation
of p42/p44 MAP kinase or p38 MAP kinase in MC3T3-E1 cells
We demonstrated that IL-1 induces phosphatidylcholine
hydrolysis by PC-PLC, resulting in the activation of PKC in
osteoblast-like MC3T3-E1 cells, and that the PKC activation then limits
IL-6 synthesis induced by IL-1 itself (9). In addition, we confirmed
that OAG, a synthetic diacylglycerol known to be an activator of PKC
(28), which alone had little effect on the synthesis of IL-6,
suppressed IL-1-induced IL-6 synthesis (Table 1). To investigate whether the limitation
of IL-1-induced IL-6 synthesis by PKC is exerted in the p42/p44 MAP
kinase- and/or p38 MAP kinase-mediating pathway(s), we examined the
effect of calphostin C, a highly potent and specific inhibitor of PKC
(29), on the IL-1-induced phosphorylation of p42/p44 MAP kinase or
p38 MAP kinase. Calphostin C, which alone weakly reduced basal
phosphorylation of p42/p44 MAP kinase, significantly enhanced
IL-1-induced p42/p44 MAP kinase phosphorylation (Fig. 5A). However, calphostin C, which by
itself hardly affected the phosphorylation of p38 MAP kinase, had
little effect on IL-1-induced p38 MAP kinase phosphorylation (Fig. 5B). We found that calphostin C suppressed IL-1-induced PKC
activation in MC3T3-E1 cells to about 10% of that in untreated cells
(Fig. 6). In the recent study (9) we
showed that calphostin C enhances IL-1-induced IL-6 synthesis in
these cells. In addition, we previously demonstrated that D-609, a
specific inhibitor of PC-PLC (30), amplifies the IL-6 synthesis induced
by IL-1 in MC3T3-E1 cells (9). Thus, we next examined the effect of
D-609 on the phosphorylation of p42/p44 MAP kinase or p38 MAP kinase in
these cells. D-609, which by itself weakly suppressed basal
phosphorylation of p42/p44 MAP kinase but hardly affected that of p38
MAP kinase, significantly amplified IL-1-induced p42/p44 MAP kinase
phosphorylation without affecting the phosphorylation of p38 MAP kinase
induced by IL-1 (Fig. 5).
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Figure 5. Effects of calphostin C or D-609 on the
IL-1-induced phosphorylation of p42/p44 MAP kinase and p38 MAP
kinase in MC3T3-E1 cells. The cultured cells were pretreated with 0.1
µM calphostin C (CalC; lanes 3 and 4), 30 ng/ml D-609
(lanes 5 and 6), or vehicle (Veh; lanes 1 and 2) for 20 min, and then
stimulated by 30 ng/ml IL-1 (lanes 2, 4, and 6) or vehicle (lanes 1,
3, and 5) for 5 min. A, p42/44 MAP kinase. Upper panel,
Antiphospho-specific p42/p44 MAP kinase antibodies. Lower
panel, Anti-p42/p44 MAP kinase antibodies. B, p38 MAP kinase.
Upper panel, Antiphospho-specific p38 MAP kinase
antibodies. Lower panel, Anti-p38 MAP kinase antibodies.
The results are representative of triplicate independent experiments.
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Effect of PKC down-regulation on IL-1-induced phosphorylation of
p42/p44 MAP kinase in MC3T3-E1 cells
In a recent study (9) we showed that the IL-1-induced IL-6
synthesis is amplified in PKC down-regulated MC3T3-E1 cells by long
term pretreatment with TPA, which is known to be a direct activator of
PKC (28). Thus, we next examined the effect of PKC down-regulation on
the IL-1-induced p42/p44 MAP kinase phosphorylation in these cells.
We previously confirmed that the binding capacity of
[3H]phorbol 12,13-dibutyrate, a PKC-activating phorbol
ester (28), in TPA (0.1 µM for 24 h)-pretreated
cells is reduced to approximately 30% of the capacity in intact cells
(31). This result suggests that PKC down-regulation truly occurs in
MC3T3-E1 cells with long term TPA treatment. The long term pretreatment
with TPA, which by itself had no effect on the phosphorylation of
p42/p44 MAP kinase, markedly increased the IL-1-induced
phosphorylation of p42/p44 MAP kinase compared with that in cells
without TPA treatment (Fig. 7).
Effects of TPA or OAG on IL-1-induced phosphorylation of p38 MAP
kinase in MC3T3-E1 cells
To elucidate whether the exogenously activated PKC affects the
phosphorylation of p38 MAP kinase, we further examined the effect of
TPA or OAG on the IL-1-induced p38 MAP kinase phosphorylation in
MC3T3-E1 cells. Either TPA or OAG, which alone had no effect on the
phosphorylation of p38 MAP kinase, failed to affect the IL-1-induced
phosphorylation of p38 MAP kinase (Fig. 8).
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Discussion
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In the present study, we demonstrated that the phosphorylation of
both p42/p44 MAP kinase and p38 MAP kinase was elicited by IL-1 in
osteoblast-like MC3T3-E1 cells. It is well known that MAP kinases are
activated by phosphorylation of threonine and tyrosine residues by dual
specificity MAP kinase kinase (10, 32). Therefore, these findings
suggest that IL-1 activates both p42/p44 MAP kinase and p38 MAP
kinase in these cells. In addition, we showed that both PD98059 and
SB203580 markedly suppressed the IL-1-induced IL-6 synthesis. We
also found that the IL-1-induced phosphorylation of p42/p44 MAP
kinase and p38 MAP kinase was truly inhibited by PD98059 and SB203580,
respectively, in these cells. Based on our present results, it is most
likely that the activations of both p42/p44 MAP kinase and p38 MAP
kinase are involved in the IL-6 synthesis induced by IL-1 in
osteoblast-like MC3T3-E1 cells.
In a recent study (9) we demonstrated that IL-1-activated PKC,
through phosphatidylcholine hydrolysis by PC-PLC, negatively regulates
IL-6 synthesis by IL-1 itself in osteoblast-like MC3T3-E1 cells. We
here confirmed that OAG suppressed the IL-1-induced IL-6 synthesis
in these cells. Thus, the detailed regulatory mechanism of PKC in the
IL-6 synthesis induced by IL-1 was investigated. We showed that
calphostin C, which hardly affected the phosphorylation of p38 MAP
kinase induced by IL-1, significantly enhanced the phosphorylation
of p42/p44 MAP kinase induced by IL-1 in these cells. Thus, it seems
that PKC activated by IL-1 inhibited the activation of p42/p44 MAP
kinase induced by IL-1 without affecting IL-1-induced activation
of p38 MAP kinase. In addition, we demonstrated that D-609 amplified
the IL-1-induced phosphorylation of p42/p44 MAP kinase without
affecting the IL-1-induced phosphorylation of p38 MAP kinase. Thus,
these results suggest that the IL-1-activated PKC through PC-PLC
suppresses the IL-1-induced activation of p42/p44 MAP kinase. Taking
our findings into account, it is most likely that p42/p44 MAP kinase,
not p38 MAP kinase, is regulated by PKC in the intracellular signaling
elicited by IL-1 in MC3T3-E1 cells, and that the attenuation of
p42/p44 MAP kinase activation by IL-1 results in suppressing the
oversynthesis of IL-6. Furthermore, we showed that the IL-1-induced
phosphorylation of p42/p44 MAP kinase was markedly increased in the PKC
down-regulated cells, suggesting that the inhibitory effect of PKC is
exerted at a point upstream from p42/p44 MAP kinase. Thus, it is
probable that our previous results showing the enhancement of
IL-1-induced IL-6 synthesis in PKC down-regulated MC3T3-E1 cells
mainly results from the amplification of p42/p44 MAP kinase activation.
In general, it is most likely that the limitation of IL-1-induced
IL-6 synthesis by PKC activation through PC-PLC is exerted at a point
upstream from p42/p44 MAP kinase in osteoblast-like MC3T3-E1 cells. It
has recently been reported that PGF2 activates p42/p44
MAP kinase through PKC in these cells (33). Thus, it is probable that
PKC possesses dual effects as an activator and a suppressor of p42/p44
MAP kinase in these cells, and these effects are dependent upon each
agonist.
In addition, we showed that TPA or OAG had little effect on
IL-1-induced phosphorylation of p38 MAP kinase in MC3T3-E1 cells.
Therefore, it seems unlikely that the PKC activation modulates
IL-1-induced activation of p38 MAP kinase in osteoblast-like
MC3T3-E1 cells. It has recently been reported that p38 MAP kinase is
involved in IL-1ß-induced IL-6 gene expression in human
fibroblast-like synoviocytes (17). From our findings, it is probable
that p38 MAP kinase also acts as a stimulative regulator in
IL-1-induced IL-6 synthesis in osteoblasts.
In conclusion, our present results strongly suggest that
IL-1-induced IL-6 synthesis is mediated via activations of both
p42/p44 MAP kinase and p38 MAP kinase in osteoblasts, and that PKC
activation negatively regulates IL-6 synthesis at a point upstream from
p42/p44 MAP kinase.
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Acknowledgments
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We are very grateful to Hidenori Kawamura and Daijiro Hatakeyama
for their skillful technical assistance.
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Footnotes
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1 This work was supported in part by a Grant-in-Aid for Scientific
Research (09671041) from the Ministry of Education, Science, Sports,
and Culture of Japan. 
Received January 11, 1999.
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Top
Abstract
Introduction
and 
) or vehicle (• and 
) for
20 min, and then stimulated by 30 ng/ml IL-1
