Inhibition by adenylyl cyclase-cAMP system of ET-1-induced HSP27
in osteoblasts
Daijiro
Hatakeyama,
Osamu
Kozawa,
Masayuki
Niwa,
Hiroyuki
Matsuno,
Kanefusa
Kato,
Norichika
Tatematsu,
Toshiyuki
Shibata, and
Toshihiko
Uematsu
Departments of Pharmacology and Oral and Maxillo-Facial Surgery,
Gifu University School of Medicine, Gifu 500 - 8705; and
Department of Biochemistry, Aichi Human Service Center, Institute for
Developmental Research, Kasugai, Aichi 480-0392, Japan
 |
ABSTRACT |
We have previously reported that endothelin-1
(ET-1) stimulates heat shock protein (HSP) 27 induction in
osteoblast-like MC3T3-E1 cells and that p38 mitogen-activated protein
(MAP) kinase acts at a point downstream from protein kinase C (PKC) in
HSP27 induction. In the present study, we investigated the effect of
the adenylyl cyclase-cAMP system on ET-1-stimulated induction of HSP27
in MC3T3-E1 cells. Dibutyryl-cAMP (DBcAMP) dose dependently
inhibited the HSP27 accumulation stimulated by ET-1. Forskolin and
cholera toxin significantly suppressed the ET-1-stimulated accumulation
of HSP27. However, dideoxyforskolin, a forskolin derivative that does
not activate cAMP, failed to suppress the ET-1-induced HSP27
accumulation. Forskolin reduced the p38 MAP kinase phosphorylation
induced by ET-1 or 12-O-tetradecanoylphorbol-13-acetate
(TPA). PGE1, an extracellular agonist that activates cAMP
production, reduced the ET-1-induced HSP27 accumulation. In addition,
the phosphorylation of p38 MAP kinase induced by ET-1 or TPA was
suppressed by PGE1. Forskolin, DBcAMP, and PGE1
suppressed the ET-1-stimulated increase in the mRNA level for HSP27.
These results indicate that the adenylyl cyclase-cAMP system has an
inhibitory role in ET-1-stimulated HSP27 induction in osteoblasts and
that the effect is exerted at the point between PKC and p38 MAP kinase
in osteoblasts.
adenosine 3',5'-cyclic monophosphate; endothelin-1; heat shock
protein 27; mitogen-activated protein kinase; protein kinase C
 |
INTRODUCTION |
WHEN CELLS ARE EXPOSED
to various stressors such as heat stress and chemical stress,
they produce heat shock proteins (HSPs; see Ref. 13). HSPs
are generally divided into high-molecular-weight HSPs and
low-molecular-weight HSPs according to apparent molecular sizes.
Low-molecular-weight HSPs, with molecular masses, from 15 to 30 kDa
such as HSP27 and
B-crystallin, have high homology in amino
acid sequences (1). High-molecular-weight HSPs are well known to act as molecular chaperones in protein folding, oligomerization, and translocation. Although the functions of low-molecular-weight HSPs are less known than those of
high-molecular-weight HSPs, it is speculated that they may act as
molecular chaperones like high-molecular-weight HSPs (1,
13). In osteoblasts, heat induces the expression of HSP27, and
estrogen reportedly facilitates the heat-induced HSP27 expression
(2, 16). Additionally, the downregulation of proliferation
has been shown to be accompanied by a transient increase of the
expression of HSP27 mRNA (2, 16). We have recently shown
that endothelin (ET)-1 stimulates HSP27 induction via the
ETA receptor and that p38 mitogen-activated protein (MAP)
kinase is involved in the HSP27 induction in osteoblast-like MC3T3-E1
cells (8). In addition, protein kinase C (PKC) activated by ET-1 acts at a point upstream from p38 MAP kinase in the HSP27 induction (8). However, the detailed regulatory mechanisms of HSP27 induction in osteoblasts and its roles have not yet been precisely clarified.
cAMP is produced from ATP by adenylyl cyclase and then causes
cAMP-dependent protein kinase (protein kinase A) activation (3). In osteoblasts, it is well recognized that the
adenylyl cyclase-cAMP system plays an important role in cell
differentiation and proliferation (17). We previously
reported that PGE1 stimulates alkaline phosphatase
activity, a marker of the mature osteoblast phenotype
(18), via cAMP production without affecting
phosphoinositide-hydrolyzing phospholipase C in osteoblast-like
MC3T3-E1 cells (6). Moreover, we have shown that
PGE1 induces interleukin-6 synthesis via protein kinase A
activation in these cells (21). Thus it is speculated that
the effects of PGE1 are exerted via the adenylyl
cyclase-cAMP system in osteoblasts. In the present study, we
investigated the effect of the adenylyl cyclase-cAMP system on the
induction of HSP27 stimulated by ET-1 in osteoblast-like MC3T3-E1
cells. Here, we report that the adenylyl cyclase-cAMP system inhibits
ET-1-stimulated HSP27 induction at the point between PKC and p38 MAP
kinase in these cells.
 |
MATERIALS AND METHODS |
Materials.
ET-1 was obtained from the Peptide Institute (Minoh, Japan).
Dibutyryl-cAMP (DBcAMP), forskolin, dideoxyforskolin, cholera toxin, 12-O-tetradecanoylphorbol-13- acetate (TPA), and
PGE1 were purchased from Sigma Chemical (St. Louis, MO).
Phosphospecific 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
(Beverly, MA). An enhanced chemiluminescence (ECL) Western blotting
detection system was obtained from Amersham Japan (Tokyo, Japan). Other materials and chemicals were obtained from commercial sources. Forskolin, dideoxyforskolin, and PGE1 were dissolved in
ethanol. TPA was dissolved in dimethyl sulfoxide. The maximum
concentrations of ethanol or dimethyl sulfoxide were 0.1%, which did
not affect immunoassay of HSP27, Northern blot analysis of HSP27, or
Western blot analysis.
Cell culture.
Cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse
calvaria (19) were maintained as previously described
(9). In brief, the cells were cultured in
-MEM
containing 10% FCS at 37°C in a humidified atmosphere of 5%
CO2-95% air. The cells were seeded in 35-mm-diameter
dishes or 90-mm-diameter dishes in
-MEM containing 10% FCS. After 5 days, the medium was exchanged for 2 ml of
-MEM containing 0.3%
FCS. The cells were used for experiments after 48 h. When
indicated, the cells were pretreated with DBcAMP, forskolin,
dideoxyforskolin, or PGE1 for 20 min. The pretreatment of
cholera toxin was performed for 12 h.
Immunoassay of HSP27.
The concentration of HSP27 in soluble extracts of cells was determined
by a sandwich-type enzyme immunoassay, as described previously
(5). The cultured cells were stimulated by ET-1 in
-MEM
for the indicated periods. The cells were then washed two times with 1 ml of PBS and frozen at
80°C for a few days before analysis. The
frozen cells on each dish were collected and suspended in 0.3 ml of
PBS, and each suspension was sonicated and centrifuged at 125,000 g for 20 min at 4°C. The supernatant was used for the
specific immunoassay of HSP27. In brief, we used an enzyme immunoassay
system that employs polystyrene balls (3.2 mm in diameter; Immuno
Chemicals, Okayama, Japan) carrying immobilized F(ab')2
fragments of antibody and the same Fab' fragments of antibody labeled
with
-D-galactosidase from Escherichia coli.
A polystyrene ball carrying antibodies was incubated either with the
purified standard for HSP27 or with an aliquot of one of the samples.
This incubation was carried out at 30°C for 5 h in a final
volume of 0.5 ml of 10 mM sodium phosphate buffer, pH 7.0, containing
0.3 M NaCl, 0.5% hydrolyzed gelatin, 0.1% BSA, 1 mM
MgCl2, and 0.1% NaN3. After being washed, each
ball was incubated at 4°C overnight with 1.5 mU of
galactosidase-labeled antibodies in a volume of 0.2 ml with 10 mM
sodium phosphate buffer, pH 7.0, containing 0.1 M NaCl, 1 mM
MgCl2, 0.1% BSA, and 0.1% NaN3. The
galactosidase activity bound to the ball was assayed using a
fluorogenic substrate, 4-methylumbelliferyl-
-D-galactoside.
Isolation of RNA and Northern blot analysis of
HSP27.
The cultured cells were stimulated by ET-1 in
-MEM for the indicated
periods. Total RNA was isolated using a QuickPrep total RNA extraction
kit (Pharmacia Biotech, Tokyo, Japan). Total RNA (20 µg) was then
subjected to electrophoresis on a 0.9% agarose-2.2 M formaldehyde gel
before being blotted on a nitrocellulose membrane. For Northern blot,
the membrane was allowed to hybridize with a cDNA probe that had been
labeled using a Multiprime DNA labeling system (Amersham,
Buckinghamshire, UK), as described previously (7). A
BamH I-Hind III fragment of
cDNA for mouse HSP27 was kindly provided by Dr. L. F. Cooper of
the University of North Carolina (2).
Western blot analysis of p38 MAP kinase.
The cultured cells were stimulated by ET-1 in
-MEM for the indicated
periods. The cells were washed two times with 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 the supernatant after
centrifugation at 125,000 g for 10 min at 4°C. SDS-PAGE was performed by the method of Laemmli (10) in 10%
polyacrylamide gel. Western blot analysis was performed as described
previously (8) by using phosphospecific p38 MAP kinase
antibodies or p38 MAP kinase antibodies, with peroxidase-labeled
antibodies raised in goat against rabbit IgG being used as secondary
antibodies. Peroxidase activity on the nitrocellulose sheet was
visualized on X-ray film by means of the ECL Western blotting detection system.
Other methods.
Protein concentrations in soluble extracts were determined using a
protein assay kit (Bio-Rad, Hercules, CA) with BSA as the standard
protein. Rat HSP27, which was used as the standard for the immunoassay,
was purified from skeletal muscle (5). The densitometric
analysis was performed using Molecular Analyst/Macintosh (Bio-Rad Laboratories).
Statistical analysis.
Each experiment was repeated three times with similar results. The data
were analyzed by ANOVA followed by the Bonferroni method for multiple
comparisons between pairs, and P < 0.05 was considered
significant. All data are presented as means ± SD of triplicate determinations.
 |
RESULTS |
Effect of DBcAMP on ET-1-induced
HSP27 accumulation in MC3T3-E1 cells.
It is well recognized that Gs functions as an intermediary
in transmembrane signaling from the receptor to adenylyl cyclase, and
the activation of adenylyl cyclase results in cAMP production (4). To clarify the role of the adenylyl cyclase-cAMP
system in ET-1-stimulated HSP27 induction in osteoblast-like MC3T3-E1 cells, we examined the effects of each direct activator of the adenylyl cyclase-cAMP system on HSP27 accumulation stimulated by ET-1.
DBcAMP, a permeable analog of cAMP, which alone had little effect on the basal level of HSP27, significantly reduced the HSP27
accumulation stimulated by ET-1 (Fig. 1).
The inhibitory effect of DBcAMP on ET-1-induced HSP27 accumulation was
dose dependent in the range between 0.1 and 3 mM. The maximum effect of
DBcAMP was observed at 3 mM, a dose that caused an ~50% reduction in the effect of ET-1 (Fig. 1).

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Fig. 1.
Effect of dibutyryl-cAMP (DBcAMP) on endothelin
(ET)-1-induced heat shock protein (HSP)27 accumulation in MC3T3-E1
cells. Cultured cells were pretreated with various doses of
DBcAMP for 20 min and then stimulated by 3 nM ET-1 ( )
or vehicle ( ) for 12 h. Each value represents the
mean ± SD of triplicate determinations. Similar results were
obtained with 2 additional and different cell preparations. *P
< 0.05 vs. ET-1 alone.
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Effects of forskolin or dideoxyforskolin on
ET-1- induced HSP27 accumulation in
MC3T3-E1 cells.
Forskolin, a direct activator of adenylyl cyclase (14)
that by itself did not affect the basal level of HSP27 inhibited ET-1-stimulated HSP27 accumulation (Fig.
2). The inhibitory effect of forskolin on
HSP27 accumulation induced by ET-1 was dose dependent in the range
between 30 nM and 50 µM. The maximum effect of forskolin was observed
at 50 µM, a dose that caused an ~84% reduction in the effect of
ET-1 (Fig. 2). However, dideoxyforskolin (10 µM), a forskolin
derivative that does not activate cAMP (15), failed to
reduce the HSP27 accumulation induced by ET-1 while forskolin (10 µM)
significantly suppressed the accumulation (Table
1).

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Fig. 2.
Effect of forskolin on ET-1-induced HSP27 accumulation in
MC3T3-E1 cells. Cultured cells were pretreated with various
doses of forskolin for 20 min and then stimulated by 3 nM ET-1
( ) or vehicle ( ) for 12 h. Each
value represents the mean ± SD of triplicate determinations.
Similar results were obtained with 2 additional and different cell
preparations. *P < 0.05 vs. ET-1 alone.
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|
Effect of cholera toxin on ET-1-induced
HSP27 accumulation in MC3T3-E1 cells.
Cholera toxin is known to be a direct activator for Gs, a
GTP-binding protein that is a stimulative regulator of adenylyl cyclase
(4). Cholera toxin significantly inhibited the
accumulation of HSP27 induced by ET-1 (Table
2).
Effect of PGE1 on
ET-1-induced HSP27 accumulation in
MC3T3-E1 cells.
PGE1 is known to stimulate cAMP production in osteoblasts
(18). In addition, we previously found that
PGE1 stimulates cAMP accumulation in MC3T3-E1 cells
(6). Thus, to investigate if a physiological agonist such
as PGE1 that stimulates cAMP production affects the
ET-1-stimulated HSP27 induction, we examined the effect of
PGE1 on ET-1-induced HSP27 accumulation. PGE1
actually significantly suppressed HSP27 accumulation stimulated by ET-1
(Table 3). PGE1 (10 µM)
caused a 26% reduction in the effect of ET-1.
Effects of forskolin, DBcAMP, or
PGE1 on the ET-1-increased
mRNA level for HSP27 in
MC3T3-E1 cells.
In a previous study (8), we have reported that the
expression level of mRNA for HSP27 was increased 2 h after the
stimulation of ET-1. We next examined the effect of forskolin on
the ET-1-induced increase in the level of mRNA for HSP27 in MC3T3-E1
cells. Forskolin, which alone had little effect on the basal level of
HSP27 mRNA, significantly suppressed the ET-1-increased level of mRNA
for HSP27 (Fig. 3A).
Furthermore, we examined the effect of DBcAMP or PGE1 on
the ET-1-induced increase in the level of mRNA for HSP27. DBcAMP or
PGE1, which alone had little effect on the basal level of
HSP27 mRNA, markedly reduced the ET-1-increased level of mRNA for HSP27
(Fig. 3B). According to the densitometric analysis, forskolin caused an ~75% reduction in the ET-1-effect. DBcAMP and
PGE1 caused an ~50 and 70% reduction, respectively (Fig.
3, A and B).

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Fig. 3.
Effects of forskolin, DBcAMP, or PGE1 on mRNA
level for HSP27 in response to ET-1 in MC3T3-E1 cells.
A: cultured cells were pretreated with 30 µM forskolin or
vehicle for 20 min and then stimulated by 3 nM ET-1 or vehicle for
2 h. B: cultured cells were pretreated with 3 mM
DBcAMP, 10 µM PGE1, or vehicle for 20 min and then
stimulated by 3 nM ET-1 or vehicle for 2 h. The cells were
harvested, and total RNA was isolated. RNA from each sample (20 µg)
was subjected to electrophoresis and blotted on a nitrocellulose
membrane. The membrane was then allowed to hybridize with the cDNA
probe for HSP27. Bands of 28S RNA are shown for reference. The
histogram shows quantitative representations of the ET-1-induced HSP27
mRNA level obtained from laser densitometric analysis of 3 independent
experiments. Each value represents the mean ± SD of triplicate
determinations. Similar results were obtained with 2 additional and
different cell preparations. *P < 0.05 vs. ET-1
alone.
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Effect of forskolin on phosphorylation of p38 MAP
kinase induced by ET-1 or TPA in
MC3T3-E1 cells.
We have recently reported that ET-1 activates p38 MAP kinase in
MC3T3-E1 cells and that p38 MAP kinase is involved in the HSP27
induction stimulated by ET-1 (8). To investigate whether or not the adenylyl cyclase-cAMP system affects ET-1-induced p38 MAP
kinase activation in these cells, we examined the effect of forskolin
on the ET-1-induced phosphorylation of p38 MAP kinase. Forskolin
markedly suppressed the ET-1-stimulated phosphorylation of p38 MAP
kinase (Fig. 4A). In a
previous study, we have shown that TPA, a PKC direct-activating phorbol
ester (11), phosphorylates p38 MAP kinase in MC3T3-E1
cells (8). Thus we investigated whether the adenylyl
cyclase-cAMP system affects p38 MAP kinase activation at a point
upstream or downstream from PKC in these cells. Forskolin significantly
suppressed the TPA-induced p38 MAP kinase phosphorylation (Fig.
4B). According to the densitometric analysis, forskolin
caused a 71% reduction in the effect of ET-1 and caused a 40%
reduction in the effect of TPA.

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Fig. 4.
Effect of forskolin on the phosphorylation of p38
mitogen-activated protein (MAP) kinase induced by ET-1 or
12-O-tetradecanoylphorbol-13-acetate (TPA) in MC3T3-E1
cells. A: cultured cells were pretreated with 30 µM forskolin or vehicle for 20 min and then stimulated by 3 nM
ET-1 or vehicle for 20 min. B: cultured cells were
pretreated with 30 µM forskolin or vehicle for 20 min and then
stimulated by 1 nM TPA or vehicle for 90 min. The extracts of cells
were subjected to SDS-PAGE against phosphospecific p38 MAP kinase
antibodies or p38 MAP kinase antibodies. The histogram shows
quantitative representations of the levels of ET-1- or TPA-induced
phosphorylation of p38 MAP kinase obtained from laser densitometric
analysis of 3 independent experiments. Each value represents the
mean ± SD of triplicate determinations. Similar results were
obtained with 2 additional and different cell preparations.
*P < 0.05 vs. ET-1 or TPA alone.
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Effect of PGE1 on p38 MAP
kinase phosphorylation induced by ET-1 or
TPA in MC3T3-E1 cells.
We next examined the effect of PGE1 on the phosphorylation
of p38 MAP kinase stimulated by ET-1 or TPA. PGE1 inhibited
the ET-1-induced p38 MAP kinase phosphorylation (Fig.
5A). In addition, PGE1 suppressed the TPA-induced p38 MAP kinase
phosphorylation (Fig. 5B). According to the densitometric
analysis, PGE1 caused a 71% reduction in the effect of
ET-1, and PGE1 almost completely reduced the effect of TPA.

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Fig. 5.
Effect of PGE1 on phosphorylation of p38 MAP
kinase induced by ET-1 or TPA in MC3T3-E1 cells.
A: cultured cells were pretreated with 10 µM
PGE1 or vehicle for 20 min and then stimulated by 3 nM ET-1
or vehicle for 20 min. B: cultured cells were pretreated
with 10 µM PGE1 or vehicle for 20 min and then stimulated
by 1 nM TPA or vehicle for 90 min. The extracts of cells were subjected
to SDS-PAGE against phosphospecific p38 MAP kinase antibodies or p38
MAP kinase antibodies. The histogram shows quantitative representations
of the levels of ET-1- or TPA-induced phosphorylation of p38 MAP kinase
obtained from laser densitometric analysis of 3 independent
experiments. Each value represents the mean ± SD of triplicate
determinations. Similar results were obtained with 2 additional and
different cell preparations. *P < 0.05 vs. ET-1 or TPA
alone.
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 |
DISCUSSION |
In a previous study (8), we have reported that
ET-1 stimulates HSP27 induction in osteoblast-like
MC3T3-E1 cells. In the present study, we showed that cholera
toxin suppressed ET-1-induced HSP27 accumulation in these cells.
Gs, a GTP-binding protein that mediates stimulative signals
from the receptor to adenylyl cyclase, induces the activation of this
enzyme (4). It is well recognized that cholera toxin
ADP-ribosylates the
-subunit of Gs and elicits continuous activation of Gs, resulting in cAMP production
(4). Thus it seems that cAMP inhibits ET-1-stimulated
HSP27 induction in osteoblast-like MC3T3-E1 cells. We next
demonstrated that forskolin inhibited HSP27 accumulation stimulated by
ET-1. Forskolin is well known as a direct activator of adenylyl cyclase
(14), and we previously found that forskolin actually
induces cAMP accumulation in MC3T3-E1 cells (9). Moreover,
dideoxyforskolin, a forskolin derivative that does not activate cAMP
(15), failed to reduce the ET-1-stimulated HSP27
accumulation. Thus our findings suggest that cAMP induced by forskolin
suppresses the HSP27 induction stimulated by ET-1 in these cells. In
addition, DBcAMP, a permeable analog of cAMP, reduced the
ET-1-increased level of HSP27. Furthermore, we showed that forskolin
and DBcAMP significantly suppressed the ET-1-increased level of mRNA
for HSP27. Therefore, based on our findings, it is most likely that the
adenylyl cyclase-cAMP system has an inhibitory role in ET-1-stimulated
HSP27 induction in osteoblast-like MC3T3-E1 cells.
In a previous study (8), we have demonstrated that
ET-1 stimulated HSP27 induction via p38 MAP kinase activation in
osteoblast-like MC3T3-E1 cells. To investigate whether or not the
adenylyl cyclase-cAMP system affects ET-1-induced p38 MAP kinase
phosphorylation in these cells, we next examined the effect of
forskolin on the p38 MAP kinase phosphorylation stimulated by ET-1.
Forskolin inhibited the phosphorylation of p38 MAP kinase induced by
ET-1. Thus our results suggest that the adenylyl cyclase-cAMP
system suppresses the ET-1-stimulated HSP27 induction at a point
upstream from p38 MAP kinase in osteoblast-like MC3T3-E1 cells. In a
previous study (20), we have shown that ET-1 stimulates
phosphatidylinositol-specific phospholipase C and
phosphatidylcholine-specific phospholipase D in osteoblast-like
MC3T3-E1 cells. Phosphatidylinositol-specific phospholipase C
hydrolyzes phosphoinositides and results in the formation of
diacylglycerol, which is well known to be a physiological activator of
PKC (11). Phosphatidylcholine-specific
phospholipase D results in the formation of phosphatidic acid from
phosphatidylcholine, and phosphatidic acid is further degraded into
diacylglycerol (12). We have previously shown that
ET-1-stimulated p38 MAP kinase activation is dependent on PKC
activation in osteoblast-like MC3T3-E1 cells (8).
Therefore, we next examined whether or not forskolin affects the
activation of p38 MAP kinase induced by TPA, a direct PKC activator in
these cells (11). Forskolin reduced the TPA-stimulated
phosphorylation of p38 MAP kinase. With our results taken into account,
it is most likely that the adenylyl cyclase-cAMP system acts as a
suppressor in the HSP27 induction stimulated by ET-1 in osteoblast-like
MC3T3-E1 cells, and the effect on HSP27 induction is exerted at the
point between PKC and p38 MAP kinase.
In a previous study (6, 21), we have shown that
PGE1 stimulates alkaline phosphatase activity via cAMP
production in osteoblast-like MC3T3-E1 cells and that PGE1
induces interleukin-6 synthesis via cAMP production. Thus we
investigated whether PGE1, a physiological agonist,
extracellulary affects the induction of HSP27 stimulated by ET-1 in
these cells. PGE1 actually suppressed the HSP27
accumulation by ET-1, forskolin, DBcAMP, and cholera toxin. The
expression of mRNA for HSP27 stimulated by ET-1 was reduced by
PGE1. Additionally, PGE1 suppressed the
phosphorylation of p38 MAP kinase induced by ET-1 or TPA in MC3T3-E1
cells. The potential effect of the adenylyl cyclase-cAMP system on
HSP27 induction stimulated by ET-1 shown in this study is summarized in
Fig. 6. According to densitometric
analysis, PGE1 caused a 71% reduction in the
phosphorylation of p38 MAP kinase induced by ET-1, whereas
PGE1 almost completely reduced p38 MAP kinase phosphorylation induced by TPA. On the other hand, DBcAMP and PGE1 caused a partial inhibition (50 and 26%,
respectively) of ET-1-induced HSP27 accumulation. Thus it seems that
other pathways, which are not inhibited by PGE1 or the
adenylyl cyclase-cAMP system, are involved in the ET-1-stimulated
HSP27 induction in osteoblast-like MC3T3-E1 cells. We previously
showed that ET-1 activates p44/p42 MAP kinase in addition to p38
MAP kinase, and its activation is not involved in ET-1- stimulated
HSP27 induction in MC3T3-E1 cells (8). Further
investigations are necessary to clarify the exact mechanism of
ET-1-stimulated HSP27 induction in osteoblasts.

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Fig. 6.
Diagram of the regulatory mechanism by the adenylyl
cyclase-cAMP system of ET-1-stimulated HSP27 induction in
MC3T3-E1 cells. , Negative modulation; PI,
phosphatidylinositol; PC, phosphatidylcholine; PLC, phospholipase C;
PLD, phospholipase D; PA, phosphatidic acid; DAG, diacylglycerol; PKC,
protein kinase C.
|
|
In conclusion, these results strongly suggest that the adenylyl
cyclase-cAMP system has an inhibitory role in ET-1-stimulated HSP27
induction in osteoblasts and that the effect is exerted at the point
between PKC and p38 MAP kinase.
 |
ACKNOWLEDGEMENTS |
We are grateful to Masaichi Miwa and Hidenori Kawamura for skillful
technical assistance.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: O. Kozawa, Dept. of Pharmacology, Gifu Univ. School of Medicine, Gifu 500-8705, Japan (E-mail: okozawa{at}cc.gifu-u.ac.jp).
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.
Received 2 March 2001; accepted in final form 3 August 2001.
 |
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