Sustained Activation of the Extracellular Signal-regulated Kinase
Pathway Is Required for Extracellular Calcium Stimulation of Human
Osteoblast Proliferation*
Zhengmin
Huang
§,
Su-Li
Cheng¶, and
Eduardo
Slatopolsky
From the
Renal Division and the ¶ Division of
Bone and Mineral Diseases, Department of Medicine, Washington
University, School of Medicine, St. Louis, Missouri 63110
Received for publication, December 4, 2000
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ABSTRACT |
Elevated levels of
[Ca2+]o in bone milieu as a result
of the resorptive action of osteoclasts are implicated in promoting proliferation and migration of osteoblasts during bone remodeling. However, mitogenic effects of [Ca2+]o have only
been shown in some, but not all, clonal osteoblast-like cells, and the
molecular mechanisms underlying [Ca2+]o-induced
mitogenic signaling are largely unknown. In this study we demonstrated
for the first time that [Ca2+]o stimulated
proliferation of primary human osteoblasts and selectively activated
extracellular signal-regulated kinases (ERKs). Neither p38
mitogen-activated protein (MAP) kinase nor stress-activated
protein kinase was activated by [Ca2+]o.
Treatment of human osteoblasts with a MAP kinase kinase inhibitor,
PD98059, impaired both basal and
[Ca2+]o-stimulated phosphorylation of ERKs and
also reduced both basal and [Ca2+]o-stimulated
proliferation. [Ca2+]o treatment resulted in two
distinctive phases of ERK activation: an acute phase and a sustained
phase. An inhibition time course revealed that it was the sustained
phase, not the acute phase, that was critical for
[Ca2+]o-stimulated osteoblast proliferation. Our
results demonstrate that mitogenic responsiveness to
[Ca2+]o is present in primary human
osteoblasts and is mediated via prolonged activation of the MAP kinase
kinase/ERK signal pathway.
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INTRODUCTION |
Extracellular calcium has been shown to control a variety of
cellular functions including secretion, cell growth, differentiation, and motility (1-3). In bone, [Ca2+]o has been
postulated to play an important role in bone remodeling. Concentrations
of [Ca2+]o fluctuate dramatically to as high as
40 mM in the local microenvironment as a result of the
resorptive action of osteoclasts (4). High concentrations of
[Ca2+]o have been suggested to stimulate
osteoblast proliferation and inhibit osteoclast resorption. Indeed,
in vitro studies showed that [Ca2+]o
and other cations stimulate proliferation in a number of
osteoblast-like cell lines (5-8). However, not all osteoblast cell
lines are responsive to [Ca2+]o (9). For example,
the effects of [Ca2+]o on proliferation of human
osteoblast cell lines are either lacking or marginal (9, 10). Varying
degrees of [Ca2+]o-stimulated proliferation among
these clonal osteoblast-like cell lines raise the interesting question
of whether calcium responsiveness exists in in vivo matured
osteoblastic cells.
Studies on [Ca2+]o responsive
osteoblast-like cell lines have so far provided only limited
information regarding the intracellular signaling mechanism underlying
[Ca2+]o stimulation of osteoblast proliferation
(3). Recent studies have shown that multiple intracellular signal
pathways in osteoblasts can be activated by
[Ca2+]o and other cations. High concentrations of
[Ca2+]o activate phospholipase C, probably
through a G-protein-coupled receptor mechanism, which then leads to
accumulation of inositol phosphates and diacylglycerol and mobilization
of intracellular calcium (8, 11-13). Activation of protein kinase C is
also required for mediating the mitogenic effects of
[Ca2+]o and cations (14). How activation of these
signal pathways leads to osteoblast proliferation is not clear.
A variety of extracellular signals have been shown to control various
cell functions such as cell proliferation and differentiation through
activation of mitogen-activated protein
(MAP)1 kinase signal pathways
(15, 16). The MAP kinase superfamily has been classified into three
subfamilies: extracellular signal-regulated kinases (ERKs),
stress-activated protein kinases/ c-Jun N-terminal kinases
(SAKs/JNKs), and p38 MAP kinase. ERKs have been best known for their
involvement in mediating intracellular signals in cell proliferation,
although members of the other two MAP kinase subfamilies have also been
reported to be capable of responding to mitogenic stimuli (17-19). In
addition, MAP kinase signal cascades have been shown to be involved in
other cellular processes such as differentiation, apoptosis, and
migration (15, 16). Although [Ca2+]o can activate
p38 MAP kinase in primary cultures of bovine parathyroid
cells2 and ERK1 in rat 1 fibroblastic cells (20), there is little information on the role and
specificity of MAP kinase signal cascades in mediating
[Ca2+]o-stimulated osteoblast proliferation.
In this study, we demonstrate that [Ca2+]o exerts
a mitogenic response in primary human osteoblasts and MG-63 cells. We
provide the first demonstration that
[Ca2+]o-stimulated cell proliferation in
osteoblasts is dependent on sustained activation of ERK1 and ERK2 but
that other MAP kinase signal pathways, p38 MAP kinase and SAPK/JNK, are
not activated by [Ca2+]o in osteoblasts.
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EXPERIMENTAL PROCEDURES |
Materials--
Unless otherwise specified, all chemical reagents
were purchased from Sigma. The tissue culture media were purchased
either from BioWhittaker, Inc. or from the Tissue Culture Support
Center (Washington University). SDS-polyacrylamide gels were purchased from Bio-Rad. The MEK1/MEK2 inhibitor PD98059 and the p38 MAP kinase
inhibitor SB203580 were purchased from Calbiochem, Inc. The protease
inhibitor mixture was purchased from Roche Molecular Biochemicals.
Cell Culture and Proliferation Assay--
Primary human
osteoblasts derived from bone chips were prepared as described
previously (21). The clonal osteoblastic cell lines (human MG-63, mouse
MC3T3-E1, and rat ST2) were routinely maintained in
-minimum
essential medium plus 10% fetal bovine serum (Life
Technologies, Inc.).
For proliferation assays using [3H]thymidine
incorporation, the cells were subcultured into 12-well plates, and the
subconfluent cells were maintained in normal culture medium for 48 h. The cells were then incubated in serum-free DMEM/Ham's F-12 medium
(1 mM Ca2+ and 0.9 mM
Mg2+) for 24 h. The cells were treated with various
agents for time periods as specified in the figure legends.
[3H]Thymidine (0.5 µCi; Amersham Pharmacia Biotech) was
added to each well and incubated in treatment medium for the last
3 h. The cells were washed once with phosphate-buffered saline and once with 5% trichloroacetic acid before 0.75 ml of 0.1 N
NaOH was added to each well to lyse the cells. The cell lysates were transferred to scintillation vials along with 10 ml of ScintiVerse (Fisher), and the radioactivity in each vial was measured in a scintillation counter. DNA synthesis measured by
[3H]thymidine incorporation was used as an index of cell proliferation.
For assessing cell proliferation by direct cell counting, cells were
subcultured into 10-cm dishes that were premarked with 0.5-cm grids.
The cells in a grid were monitored using an inverted light microscope
and photographed through a CCD camera at 0-, 24-, 48-, and 72-h
time points. The cell numbers were measured using at least three grids
that contain similar cell numbers at the starting time points.
Inhibitor Experiments--
For inhibition of MEK, PD98059 was
added to DMEM/Ham's F-12 medium at a final concentration of 50 µM 1 h before calcium treatment. For the removal of
PD98059, cells treated with PD98059 were washed three times for 1 h each with serum-free DMEM/Ham's F-12 medium. For inhibition of p38
MAP kinase, SB 203580 was added to DMEM/Ham's F-12 medium at a final
concentration of up to 10 µM 1 h before calcium treatment.
Cell Extract Preparation--
At the end of the treatment
period, media were removed immediately, and the cells in 10-cm dishes
were lysed by the addition of 0.5 ml of the kinase extraction buffer
(20 mM HEPES, 150 mM NaCl, 2 mM
Na3VO4, 1 mM NaF, 5 mM
EDTA, 10% glycerol, 1% Triton X-100, and protease inhibitor mixture,
1 tablet/10 ml of buffer). The cells were detached from dishes by
scraping, and the cell lysates were collected in 1.5-ml microcentrifuge
tubes. After a 5-min centrifugation at 12,000 rpm, the supernatants
were collected and stored at
80 °C. The protein concentrations of
the samples were determined by Bradford protein assay (Bio-Rad).
Analysis of MAP Kinase Activation--
Activation of various MAP
kinases (ERK1/2 MAP kinases, p38 MAP kinase, and SAPK/JNK) has been
shown to occur through dual phosphorylation of threonine and tyrosine
residues by upstream MAP kinase kinases. The dual phosphorylation
sequences are TEY for ERK1/2, TGY for p38, and TPY for SAPK/JNK. The
levels of dual phosphorylation at these positions in both control and
treated samples were measured using PhosphoPlus antibody kits (New
England Biolabs, Inc.) following the manufacturer's instructions.
Phosphorylation of specific MAP kinases was detected by
affinity-purified antibodies recognizing specific dual phospho-peptide
sequences present in these MAP kinases. As a control, antibodies
recognizing MAP kinases independent of their phosphorylation status
were used to determine the total amount of individual MAP kinases
present in the samples. Briefly, protein samples were size-fractionated
by 10% SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes (Bio-Rad) by electroblotting. The membranes
were blocked in 5% nonfat dry milk for 1 h at room temperature
followed by an overnight incubation with the primary antibody at
4 °C in Tris-buffered saline (10 mM Tris, pH 8.0, 150 mM NaCl) containing 0.1% Tween 20 (TBS-T). The membranes
were washed three times in TBS-T and incubated with the appropriate
secondary antibody conjugated to horseradish peroxidase for 1 h at
room temperature. After extensive washing in TBS-T, membranes were
subject to a chemiluminescence-based detection assay (New England
Biolabs, Inc.).
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RESULTS |
Proliferative responses to [Ca2+]o
stimulation have been demonstrated in a number of osteoblastic cell
lines (6, 9, 10, 22). However, varying degrees of calcium
responsiveness were reported among various human and mouse osteoblastic
cell lines (5, 9, 10). Thus it raises the interesting question of
whether calcium responsiveness is an intrinsic property of osteoblastic
cells. To address this question, we assessed the effects of
[Ca2+]o on the proliferation of primary human
osteoblasts. Treatment of quiescent primary culture of human
osteoblasts with 5 mM Ca2+ resulted in a 5-fold
stimulation of DNA synthesis over the control value (1 mM
Ca2+) (Fig. 1). To compare
the degree of calcium responsiveness of the primary human osteoblast
cells with those of clonal osteoblast-like cells, we also determined
[Ca2+]o-stimulated cell proliferation of human
MG-63 cells, mouse MC3T3-E1 cells, and rat ST2 cells. The proliferative
responses to [Ca2+]o stimulation were found to be
similar among all of these osteoblastic cells (Fig. 1).

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Fig. 1.
[Ca2+]o stimulates
proliferation of human, rat, and mouse osteoblasts. Primary human
osteoblasts (HOB), human MG-63 cells, mouse MC3T3-E1 cells,
and rat ST2 cells were treated with 1 mM (open
bars) or 5 mM calcium (filled bars) for
48 h, and [3H]thymidine incorporation was used as an
index of cell proliferation.
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Demonstration of [Ca2+]o-stimulated proliferation
in primary human osteoblastic cells indicates that calcium
responsiveness is an intrinsic property of human osteoblast cells and
supports the hypothesis that [Ca2+]o may play a
critical role in the bone remodeling process in vivo. The
degrees of mitogenic response observed in our studies were very similar
among various cell lines across different species, suggesting that the
differences in calcium responsiveness reported in the literature may be
the results of the different experimental conditions and the specific
cell lines established (5, 7, 9, 10). Because the human osteoblastic
cell line MG-63 responded to [Ca2+]o in a manner
similar to that of primary human osteoblasts, we therefore chose to use
MG-63 cells for all of the subsequent experiments.
[Ca2+]o-stimulated proliferation in MG-63 cells
was dose-dependent (1-10 mM) with maximum
stimulation at 5 mM (Fig. 2A). The mitogenic effect of
[Ca2+]o was also time-dependent
(3-72 h) with increments observed at 24-72 h in cells treated with 5 mM [Ca2+]o (Fig. 2B).
Interestingly, no differences in [3H]thymidine
incorporation were found in the 3-h treatment (day 0) between 1 and 5 mM [Ca2+]o-treated groups, suggesting
that prolonged treatment of [Ca2+]o was required
for stimulation of cell proliferation.

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Fig. 2.
Effects of time, dose, and various cations on
MG-63 cell proliferation. A, MG-63 cells were incubated
for 48 h in DMEM/Ham's F-12 medium containing 1, 2, 5, or 10 mM calcium. B, MG-63 cells were incubated with
DMEM/Ham's F-12 medium containing 1 or 5 mM calcium for 3, 24, 48, or 72 h. C, MG-63 cells were incubated for
48 h with DMEM/Ham's F-12 medium containing 1 mM
calcium (open bars) or 5 mM calcium, 100 µM gadolinium, 300 µM neomycin, or 100 µM nickel (filled bars).
[3H]Thymidine incorporation was used as an index of cell
proliferation.
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To rule out the possibility that calcium-stimulated increase of
[3H]thymidine incorporation was due to unscheduled DNA
synthesis such as DNA repair following damage, we assessed the effects
of extracellular calcium on osteoblast proliferation by directly measuring osteoblast cell number. MG-63 cells were treated with 1 or 5 mM calcium for 24, 48, and 72 h, and the numbers of
osteoblast cells in the 5 mM calcium-treated group
were progressively further increased by 1.5-, 2.4-, and 4.1-fold,
whereas the number of osteoblast cells treated with 1 mM
calcium was only marginally increased by 1.1-, 1.3-, and 1.7-fold (Fig.
3). Thus extracellular calcium clearly
stimulates osteoblast proliferation by enhancing DNA synthesis as well
as increasing cell number. This result validates the use of
[3H]thymidine incorporation as an index for cell
proliferation.

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Fig. 3.
[Ca2+]o progressively
increases osteoblast cell number. MG-63 cells were incubated for
24, 48, and 72 h in DMEM/Ham's F-12 medium containing 1 mM (circle) or 5 mM
(square) calcium. The cell numbers were counted at each time
points as described under "Experimental Procedures."
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Cations other than calcium have been reported to be able to activate
cell proliferation in a number of osteoblast-like cell lines. We
therefore assessed the ability of several cations (Gd3+,
Ni2+, and neomycin) to elicit mitogenic responses in MG-63
cells (Fig. 2C). Gd3+ (100 µM), a
known agonist for the cloned calcium-sensing receptor (CaR), was able
to stimulate cell proliferation to almost the same degree as that
induced by Ca2+. Gd3+ is a non-cell-permeable
cation, suggesting that the entry of the cation into the cells might
not be required for stimulation of cell proliferation (23). In
contrast, Ni2+ treatment (100 µM) did not
induce a mitogenic response in MG-63 cells. Ni2+ ion is not
an agonist for the cloned CaR but is a known agonist for the
ranyodine-like receptor present in the osteoclasts (3, 24),
suggesting that a ranyodine-like receptor was probably not involved in
mediating cation-induced mitogenesis in osteoblasts. The addition of
neomycin (300 µM), a known agonist for CaR, also weakly
stimulated cell proliferation. Thus these combined data are consistent
with the hypothesis that [Ca2+]o stimulates
osteoblast cell proliferation through the cloned CaR or other
calcium-sensing receptor(s) with similar cation specificity (5, 13,
25).
Various extracellular mitogenic signals such as growth factors and
cytokines have been shown to stimulate cell proliferation through ERK,
p38, or SAPK/JNK MAP kinase signal pathway (17-19). To determine
whether [Ca2+]o-stimulated osteoblast
proliferation was mediated by any of these MAP kinase signal pathways,
we first assessed whether any of the MAP kinases were activated by
[Ca2+]o. We took advantage of the fact that
activation of all three types of MAP kinases occurs through dual
phosphorylation of threonine and tyrosine residues that can be
recognized by antibodies specific for the phospho-proteins. The
quiescent MG-63 cells were treated with [Ca2+]o
for various time periods, and activation of ERK1 and ERK2 was measured
by Western blotting analysis. At the basal state (1 mM
calcium), we detected low but measurable phosphorylation of ERK1 and
ERK2 using a phospho-antibody that recognizes the dual phosphorylated,
active form of ERK1 and ERK2 (Fig. 4,
top left panel). Thus it indicated that even at 1 mM calcium, low levels of activated ERK1 and ERK2 were
present and might contribute to low but measurable basal proliferation.
However, a rapid increase (within 5 min) in phosphorylation over basal
levels was observed for both ERK1 and ERK2 following 5 mM
calcium treatment. The phosphorylation levels of the ERK1 and ERK2
peaked after 5 min of calcium stimulation and gradually declined with
time. However, even at 60 min, phosphorylation of ERK1 and ERK2 was
still significantly higher than basal levels. We thus examined whether
sustained ERK phosphorylation occurred with longer exposure to elevated
[Ca2+]o (2, 6, 24, 48 h). The increases in
phosphorylation of ERK1 and ERK2 were maintained for up to 48 h
(Fig. 4, top right panel), indicating that
[Ca2+]o stimulation resulted in sustained
activation of ERK1 and ERK2. This sustained activation was consistent
with our finding that [Ca2+]o-stimulated
proliferation required prolonged treatment of
[Ca2+]o.

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Fig. 4.
[Ca2+]o stimulates
phosphorylation of ERK 1/2 MAP kinases. Western blot analysis was
performed with extracts from cells treated with 1 or 5 mM
calcium for specified times using an antibody against the active form
(phosphorylated form) of ERK 1/2 MAP kinases (top panel) or
an antibody recognizing both the active and inactive forms of ERK 1/2
MAP kinases (bottom panel).
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The increase in phosphorylation of ERK MAP kinases, especially in the
sustained phase, could be due to three mechanisms: 1) an increase in
the percentage of phosphorylated proteins in the existing ERK MAP
kinase pool; 2) an increase in overall expression levels of ERK MAP
kinases without altering the percentage of phosphorylation; or 3) a
combination of the two. Western blotting analysis using the antibody
recognizing all forms of ERK1 and ERK2 showed that the total amounts of
ERK1 and ERK2 were similar at all time points (Fig. 4, bottom
panels), indicating that protein expression was not significantly
altered. Thus the increases of phosphorylation in both the acute and
the sustained phases were due to the increase in the percentage of
phosphorylation in the existing ERK MAP kinases rather than as the
consequence of an increase in the overall expression levels of ERK MAP kinases.
The ability of Gd3+ to mimic calcium in stimulating
osteoblast proliferation raised the question of whether
Gd3+ can also stimulate ERK phosphorylation. To
address this, MG-63 cells were treated with 100 µM
Gd3+ for various time periods. Gd3+ not only
stimulates acute phosphorylation of the ERKs but also induced sustained
activation of the ERKs up to 24 h (Fig.
5). This pattern of activation is similar
to that observed with calcium stimulation, suggesting that two
different ions may use a common mechanism for activation of ERKs.

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Fig. 5.
[Gd3+] induces
acute and sustained phosphorylation of ERK1/2 MAP kinases in MG-63
cells. Western blot analysis was performed with extracts from
cells treated with 0 or 100 µM gadolinium for specified
times using an antibody against the active form (phosphorylated form)
of ERK 1/2 MAP kinases (top panel) or an antibody
recognizing both active and inactive forms of ERK 1/2 MAP kinases
(bottom panel). Cell extracts from cells treated with 1 or 5 mM calcium for 24 h were used as negative and positive
controls (lanes 13 and 14).
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To assess whether other MAP kinases such as p38 MAP kinase and SAPK/JNK
were activated by [Ca2+]o, we assessed
phosphorylation of p38 MAP kinase and SAPK/JNK using phospho-antibodies
against phosphorylated peptides derived from p38 MAP kinase and the
SAPK/JNK, respectively. We found that p38 MAP kinase was not
phosphorylated at either the basal or
[Ca2+]o-stimulated conditions, whereas sufficient
protein expression was detected (Fig. 6,
A and B). Similarly, no phosphorylation of
SAPK/JNKs was observed when cells were treated with 5 mM
[Ca2+]o, whereas expression of SAPK/JNKs was
readily seen (Fig. 6, C and D). This result
demonstrated that [Ca2+]o did not activate either
p38 MAP kinase or SAPK/JNK in MG-63 cells and that neither p38 MAP
kinase nor SAPK/JNK was likely to play any role in
[Ca2+]o-stimulated osteoblast cell
proliferation.

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Fig. 6.
[Ca2+]o does not
stimulate phosphorylation of p38 and SAPK/JNK MAP kinases. Western
blot analysis was performed with extracts from cells treated with 1 or
5 mM calcium for specified times using an antibody
recognizing both the active and inactive forms of p38 and SAPK/JNK MAP
kinases (A and C) and an antibody against the
active form (phosphorylated form) of p38 and SAPK/JNK MAP kinases
(B and D). Negative and positive control cell
extracts were in lanes 1 and 2.
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ERK1 and ERK2 are known to be activated by the upstream enzyme MEK1 and
MEK2, and such activation can be blocked by the MEK1/MEK2 inhibitor
PD98059 (26-28). To test whether PD98059 could block activation of
ERK1 and ERK2 by [Ca2+]o, we treated MG-63 cells
with 50 µM PD98059 for 1 h before calcium
stimulation. PD98059 treatment not only markedly reduced basal
phosphorylation of ERK1 and ERK2 but also completely eliminated
[Ca2+]o-stimulated phosphorylation (Fig.
7).

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Fig. 7.
Inhibition of MEK by PD98059 blocks both
basal and [Ca2+]o-induced phosphorylation of ERK
1/2 MAP kinases MG-63 cells were incubated with PD98059 (50 µM) for 1 h, and then the medium
calcium concentration was adjusted to either 1 mM or 5 mM followed by incubation at 37 °C for additional 10 min. Western blot analysis was performed with extracts from MG-63
cells with various treatments using an antibody against the active form
(phosphorylated form) of ERK 1/2 MAP kinases (top panel) or
an antibody recognizing both the active and inactive forms of ERK 1/2
MAP kinases (bottom panel).
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To address the question of whether activation of the MEK/ERK signal
cascade (phosphorylation of ERK1 and ERK2) was functionally important
for [Ca2+]o-stimulated proliferation, we measured
[Ca2+]o-stimulated [3H]thymidine
incorporation in the presence and absence of PD98059 (Fig.
8). PD98059 treatment not only severely
impaired [Ca2+]o-stimulated proliferation but
also markedly reduced basal cell proliferation (Fig. 8). This result
showed that ERK MAP kinases were important in both
[Ca2+]o-stimulated and basal cell proliferation.
It was consistent with the effects of PD98059 on phosphorylation of
ERK1 and ERK2 (Fig. 7), where both basal and
[Ca2+]o-stimulated phosphorylation of ERK1 and
ERK2 were blocked by PD98059. This result indicated that there might be
a direct association of ERK1 and ERK2 activation with cell
proliferation in MG-63 cells.

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Fig. 8.
Inhibition of MEK by PD98059 severely impairs
both basal and [Ca2+]o-stimulated proliferation
of MG-63 cells. MG-63 cells were incubated in the absence
(Control) or the presence of 50 µM PD98059
(PD98059 and Recovery) for 1 h, and then the
calcium concentration in the medium was adjusted to 1 or 5 mM. For the control and PD98059 groups, cells were
incubated at 37 °C for 24 h before harvest. For the recovery
group, after 24-h treatment with PD98059 in 1 or 5 mM
calcium, cells were washed three times for 1 h each with
DMEM/Ham's F-12 medium (1 mM calcium) to remove PD98059.
The cells were then treated with 1 mM (open
bars) or 5 mM (filled bars) calcium for
another 24 h before harvest. [3H]Thymidine
incorporation was used as an index of cell proliferation.
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To rule out the possibility that the decrease in cell proliferation was
due to cell death as a result of the potential cytotoxicity of PD98059,
we carried out a recovery experiment, in which MG-63 cells were first
treated with PD98059 for 24 h and then washed three times for
1 h each with normal DMEM/Ham's F-12 medium containing 1 mM Ca2+ to remove PD98059. The cells were then
treated with 1 or 5 mM Ca2+ for an additional
24 h (Fig. 8). As shown in Fig. 8, 5 mM calcium treatment of MG-63 cells following the removal of PD98059 resulted in a
~3-fold increase in cell proliferation over that of 1 mM calcium (recovery group). The fold induction was similar to the fold
induction in the control group. Regaining calcium responsiveness after
removal of PD98059 indicated that MG-63 cells were still functionally
viable after 24 h of PD98059 treatment. Thus blocking phosphorylation of ERK1 and ERK2 with PD98059 suppressed both basal and
[Ca2+]o-stimulated osteoblast cell proliferation,
indicating that the phosphorylation of ERK1 and ERK2 was critical for
these two processes.
To directly assess whether p38 MAP kinase activity was required for
calcium-stimulated proliferation, we incubated MG-63 cells with a
potent p38 MAP kinase inhibitor, SB203580, which has an in
vitro IC50 of ~0.6 µM (29, 30). Both 1 and 10 µM concentrations of SB203580 failed to inhibit
calcium-stimulated osteoblast proliferation (Fig.
9). This result further confirmed that
p38 MAP kinase was not critical for calcium-stimulated osteoblast
proliferation.

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Fig. 9.
Inhibition of p38 MAP kinase by SB203580 does
not block [Ca2+]o-stimulated proliferation of
MG-63 cells. MG-63 cells were incubated in DMEM/Ham's F-12 medium
containing 1 mM (open bars) or 5 mM
(filled bars) calcium and in the absence
(Control) or the presence of SB203580 (1 and 10 µM) for 24 h. [3H]Thymidine
incorporation was used as an index of cell proliferation.
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A number of recent studies suggested that sustained MAP kinase
activation was required for cellular functions such as cell proliferation and differentiation (31-34). To assess whether the acute
or the prolonged activation of ERK1/2 was responsible for mediating
mitogenic effects of [Ca2+]o, we first tested
whether transient exposure to high calcium was sufficient to cause
sustained activation of ERK1/2 and/or increase cell proliferation.
MG-63 cells were transiently exposed to 5 mM
[Ca2+]o for 1 h and then incubated in normal
growth medium (1 mM [Ca2+]o) for an
additional 1 or 23 h. The cells were then either harvested for
assessing ERK activation or treated with [3H]thymidine to
measure cell proliferation. One hour after removal of high calcium
medium, ERK phosphorylation levels were reduced back to the basal
level, and subsequent incubation with 1 mM calcium up to
24 h did not result in reactivation of the ERKs (Fig.
10). Transient exposure to 5 mM [Ca2+]o (1 or 2 h) did not
result in increase in [3H]thymidine incorporation at
24 h either (Fig. 11). These
results indicate that transient exposure to high calcium causes acute activation of ERKs but does not lead to sustained activation of ERKs.
Acute activation of ERK1/2 by transient calcium treatment was not
sufficient to stimulate osteoblast proliferation. This result suggested
that the continuing presence of high calcium is required for sustained
activation of ERKs and stimulation of osteoblast proliferation.

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Fig. 10.
Transient calcium stimulation induces acute
activation but not sustained activation of ERK 1/2 MAP kinases.
Western blot analysis was performed with extracts from cells treated
with 1 or 5 mM calcium for specified time points using an
antibody against the active form (phosphorylated form) of ERK 1/2 MAP
kinases (A) or an antibody recognizing both active and
inactive forms of ERK 1/2 MAP kinases (B). Lanes
1-8, cell extracts from cells that were treated with 1 or 5 mM calcium for 5 min, 1 h, 2 h, and 24 h.
Lanes 9-12, cell extracts from cells that were treated with
1 or 5 mM calcium for 1 h and then switched to 1 or 5 mM calcium for 1 or 23 h.
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Fig. 11.
Transient calcium stimulation does not
induce proliferation of MG-63 cells. MG-63 cells were incubated in
1 mM (open bars) or 5 mM
(filled bars) calcium at 37 °C for 24 h (24 hr). For transient calcium treatment, cells were treated with 1 or
5 mM calcium for 1 h or 2 h and then switched to
1 mM calcium for 24 h (1 hr and 2 hr). [3H]Thymidine incorporation was used as an
index of cell proliferation.
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To directly test whether sustained activation of ERK1/2 as the result
of prolonged treatment of high calcium is required for calcium-stimulated osteoblast proliferation, we first treated MG-63
cells with 5 mM [Ca2+]o for 1 h
and then added PD98059 to the medium (Fig. 12). The delayed treatment of cells
with PD98059 suppressed the proliferative effect of
[Ca2+]o. The degree of suppression was similar to
that obtained by pretreatment with PD98059. (PD98059 was added 1 h
prior to addition of 5 mM Ca2+.) Thus it
confirmed that acute activation of ERK1 and ERK2 was not sufficient for
[Ca2+]o-stimulated proliferation. It also
demonstrated that prolonged treatment with high calcium in the absence
of sustained activation of ERKs was not sufficient for stimulation of
osteoblast proliferation, which indicated that prolonged activation of
ERK1 and ERK2 was critical for [Ca2+]o-stimulated
osteoblast proliferation.

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Fig. 12.
Delayed inhibition of MEK by PD98059
suppresses [Ca2+]o-stimulated proliferation of
MG-63 cells. MG-63 cells were incubated in 1 mM
(open bars) or 5 mM (filled bars)
calcium at 37 °C for 24 h. For the pretreatment group, cells
were treated with 50 µM PD98059 in DMEM/Ham's F-12
medium (1 mM calcium) for 1 h before the calcium
concentration in the medium was adjusted to 1 or 5 mM. For
the post-treatment group, PD98059 (50 µM) was added to
DMEM/Ham's F-12 medium after cells were treated with 1 or 5 mM calcium for 1 h. [3H]Thymidine
incorporation was used as an index of cell proliferation.
|
|
 |
DISCUSSION |
Successful bone remodeling requires balanced rates of bone
formation and bone resorption. The rates of bone formation are dependent on the number of functionally active osteoblasts. It is thus
of prime importance to identify the extracellular factors in the bone
milieu that regulate the proliferation of osteoblasts. In this study,
we demonstrated that [Ca2+]o is indeed such an
extracellular factor that stimulated proliferation of primary human
osteoblast cells and clonal osteoblast cell lines across a number of
species. We also provided clear evidence for the first time that
[Ca2+]o stimulation of human osteoblasts resulted
in both an acute phase and a sustained phase of ERK1 and ERK2
activation, which were through dual phosphorylation of critical
threonine and tyrosine residues. The sustained activation was required
for proliferative responses, whereas the acute activation was not sufficient. We found that although p38 MAP kinase and SAPK/JNK were
present in MG-63 cells, they were not activated by
[Ca2+]o stimulation. Thus our studies suggest
that the ability to sense [Ca2+]o is present in
in vivo matured human osteoblasts, supporting the hypothesis
that [Ca2+]o plays an important role in the bone
remodeling process. Our findings also establish a molecular and
cellular mechanism for [Ca2+]o-stimulated
osteoblast proliferation. The mitogenic effects of
[Ca2+]o are mediated through sustained activation
of specific ERK1 and ERK2 MAP kinases. Our results do not support the
possibility that other MAP kinases such as p38 MAP kinase and SAPK/JNK
are involved in [Ca2+]o-stimulated osteoblast proliferation.
The discovery that ERK1 and ERK2 MAP kinases play a key role in
[Ca2+]o-stimulated osteoblast proliferation
raises an interesting question regarding the molecular nature of the
upstream signaling cascades leading toward activation of ERK1 and ERK2.
Growth factor receptors and G-protein-coupled receptors have all been
shown to activate ERK MAP kinase signal cascades through a number of mechanisms (15, 35, 36). Several studies provided molecular evidence
that the cloned G-protein-coupled CaR was present in osteoblastic
cells, suggesting that the changes in [Ca2+]o may
be sensed by the CaR, which then activates intracellular signal
cascades (5, 6, 37). A very recent study demonstrated unambiguously
that the mRNA and protein of the CaR were present in MG-63 cells
and that the CaR was actively coupled to a K+ channel in
MG-63 cells (13). Thus this evidence clearly resolved the controversial
issue of the presence of the CaR in osteoblast cells and supported the
hypothesis that the CaR is a possible candidate for mediating
[Ca2+]o-stimulated osteoblast proliferation.
Cation specificity displayed by MG-63 cells in this study is
functionally consistent with cation specificity of the cloned
G-protein-coupled CaR (1, 38). However, the critical issues of whether
the cloned CaR is the only calcium sensor in MG-63 cells and whether it
plays any functional role in mediating
[Ca2+]o-stimulated osteoblast proliferation
remain unsolved (3, 9). More direct functional studies are needed to
firmly establish a role for the CaR as an upstream component in
[Ca2+]o-stimulated osteoblast proliferation.
Although acute activation of MAP kinase signal cascades by
extracellular signals have been established in many cell systems, sustained activation of MAP kinases has only been reported in a limited
number of studies, all of which indicate that the sustained activation,
but not the acute activation, plays a more critical role in cell
proliferation, differentiation, and migration as well as gene
expression (31, 33, 39). This study establishes an important role for
sustained activation of ERK1 and ERK2 in mediating the mitogenic
effects of [Ca2+]o on osteoblast cell
proliferation. Further studies examining the molecular mechanism
underlying sustained activation will provide insight regarding how
[Ca2+]o regulates osteoblast proliferation and
the bone remodeling process.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Adriana Dusso, Alex Brown, and
Michael Rauchman for helpful discussion during the course of this study
and for critical review of this manuscript.
 |
FOOTNOTES |
*
This work was supported in part by NIDDK, National
Institutes of Health Grants DK-09976, DK-30178, and DK-07126 (to
E. S.).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 correspondence should be addressed: Washington University
Medical School, Dept. of Medicine, Renal Division, 660 South Euclid
Ave. Box 8126, St. Louis, MO 63110. Tel.: 314-362-8246; Fax:
314-362-8237; E-mail: zhuang@im.wustl.edu.
Published, JBC Papers in Press, April 5, 2001, DOI 10.1074/jbc.M010921200
2
A. Brown, personal communication.
 |
ABBREVIATIONS |
The abbreviations used are:
MAP, mitogen-activated protein;
ERK, extracellular signal-regulated kinase;
SAPK, stress-activated protein kinase;
JNK, c-Jun N-terminal kinase,
CaR, calcium sensing receptor;
MEK, MAP kinase kinase;
DMEM, Dulbecco's modified Eagle's medium.
 |
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