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INTRODUCTION |
Bone adapts to mechanical and physiological stress by a unique
form of tissue replacement contained within discrete structures defined
as basic multicellular units (2). Within each of these units, the
actual process of bone removal is carried out by osteoclasts; replacement bone matrix is synthesized and mineralized by cells of
stromal origin, osteoblasts. Examination of resorbing sites in
developing skeletal tissues indicates that many of the cells are
apoptotic (3). Thus, there is evidence of DNA fragmentation in
osteoclasts, osteoblasts, and osteocytes (4, 5). In contrast, in mature
skeletal tissues, only about 1-2% of all bone cells are dying or
dead. In both the developing and mature skeleton, most of the apoptotic
cells are confined to bone remodeling sites or locales of high bone
turnover (5-8).
How osteoclasts communicate with and regulate the life history of other
cells of the basic multicellular unit is a topic of intense debate. It
is clear that osteoclast differentiation and activation are dependent
on paracrine signals received from stromal cells in the multicellular
unit (9). Within the past four years, chemical modulators of these
processes have been identified, and recent evidence indicates that
growth factors provide survival signals that result in bone cell
proliferation and depression of the apoptotic process (5, 6, 10). In
addition, it has been demonstrated that a number of pharmacological
agents can induce osteoblast apoptosis in vitro (5, 10-12).
Surprisingly, however, little is known of events that promote
osteoblast death in situ.
Although the resorption process may generate agents that stimulate
osteoblast proliferation, it is probable that products of the resorbing
bone may also stimulate bone cell death. Recent work from this
laboratory has clearly demonstrated that one of the ions present in the
bone matrix, inorganic phosphate (Pi), induces apoptosis of
cultured human osteoblasts and chondrocytes (1, 13). Since
Ca2+ as well as Pi are released from the bone
apatite lattice during the resorption process, the possibility exists
that Ca2+ may influence Pi-mediated bone cell
apoptosis. To test the hypothesis that this ion pair may trigger
the death program, we examine the effect of Pi and
Ca2+ on human osteoblast-like cells. We ask the questions,
Can Ca2+ modulate Pi-induced cell death, and,
if so, is death mediated by apoptosis? Using a cell culture system, we
demonstrate that Ca2+ accentuates the apoptogenic effect of
Pi. In addition, we provide evidence for the involvement of
mitochondria and intracellular Ca2+ in the apoptotic
pathway activated by the ion pair.
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MATERIALS AND METHODS |
Cell Culture--
Specimens of human bone were obtained during
dental surgery performed at the Hospital of the University of
Pennsylvania and during spinal surgery performed at the Children's
Hospital of Philadelphia (Philadelphia, PA). Ages of the samples ranged
from 9 to 32 years. The bone was chopped into very small pieces using a
pair of rongeurs. The pieces were then digested in 10 ml of bacterial
collagenase (4.6 mg/ml) (Sigma) in Ca2+- and
Mg+-free Hanks' balanced salt solution for 1 h at
37 °C in a shaker bath. The supernatant was discarded, and the bone
fragments were placed into cell culture dishes (Corning Glass, Corning,
NY) containing Dulbecco's modified eagle's medium
(DMEM)1 (Life Technologies,
Inc.) supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, and antibiotics. The Pi
concentration of this medium was 0.9 mM, and the
Ca2+ concentration ([Ca2+]e) was 1.8 mM. The cultures were maintained at 37 °C in a sterile
incubator, and the medium was changed every day.
Cell migration from the bone fragments was monitored daily by light
microscopy. After a period of 3-6 weeks, osteoblast-like cells grew
out from the explant. When confluent, cells were released from the
tissue culture dishes by a brief treatment with 0.25% trypsin and
0.1% bacterial collagenase (Sigma) in Hanks' balanced salt solution.
Cells were collected and replated at a density of 140 cells/mm2. Secondary cultures were maintained in DMEM
supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 5 mM
-glycerophosphate, and
antibiotics. After 48 h, fresh ascorbate (10 µg/ml) was added to
the medium. Cells were fed fresh ascorbate at every media change. The
osteogenic characteristics of the cells were confirmed by reverse
transcription-polymerase chain reaction using probes for Cbfa-1,
osteocalcin, osteonectin, and type I collagen (as described in Meleti
et al. (1)).
We also examined the effect of the ion pair on MC-3T3-E1
cells, a cell line that, in culture, recapitulates each of the major steps in osteoblast maturation (14). These cells were grown to
confluence in DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and 25 µg/ml ascorbate
as described above. After 7 days in culture, the osteogenic
characteristics of the cells were confirmed by reverse
transcription-polymerase chain reaction using probes for Cbfa-1,
osteocalcin, osteonectin, and type I collagen (see above).
Induction of Cell Death--
Osteoblast viability was assessed
as a function of the Pi concentration and
[Ca2+]e as well as treatment time. Pi
was added to the medium in the form of sodium phosphate. The addition
of 2, 4, and 6 mM sodium phosphate resulted in a final
medium Pi concentration of 3, 5, and 7 mM.
Ca2+ was added as calcium chloride. The addition of 0.1, 0.5, and 1 mM Ca2+ resulted in final medium
Ca2+ concentrations of 2.0, 2.4, and 2.9 mM
Ca2+, respectively. In a parallel experiment, the effect of
the Na-Pi transport inhibitor, phosphonoformic acid (PFA),
on ion pair-induced cell death was also evaluated. In addition, we
determined whether the Ca2+ channel blockers (verapamil,
nifedipine, lanthanum chloride, and gadolinium chloride) could modulate
Ca2+- and Pi-dependent apoptosis.
All of these agents were purchased from Sigma. In each case, cell death
was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.
MTT Assay--
The MTT assay is based on the ability of
mitochondria in live cells to oxidize thiazolyl blue, a tetrazolium
salt (MTT; Sigma)), to an insoluble blue formazan product. Cells
were treated with the agents for the indicated time periods, washed,
and then incubated with MTT (120 µg/ml) at 37 °C for 3 h. The
reagent was removed, and 400 µl of 0.04 M HCl in
isopropanol was added to each well. The optical density of the solution
was read at 595 nm in an enzyme-linked immunosorbent assay
(enzyme-linked immunosorbent assay) plate reader (15). Since the
generation of the blue product is proportional to the mitochondrial
dehydrogenase activity, the decrease in the absorbance at 595 nm
provided a direct measurement of cell death.
Detection of Apoptosis--
Although we have previously
demonstrated that Pi induced osteoblast apoptosis (1), it
was important to determine whether the ion pair kills bone cells by
activating apoptosis. Three different approaches were employed.
The TUNEL assay takes advantage of the fact that during apoptosis,
nuclear endonucleases cleave linker DNA into fragments of multiples of
~200 base pairs. The cell cultures were treated with the ion pair,
and the fragmented nucleotide ends were labeled using a Klenow FragEL
kit (Oncogene Research products, Cambridge, MA). Untreated osteoblasts
were used as controls. Cells were then treated with proteinase K (20 mg/ml) at room temperature for 15 min. We have previously established
that the duration of proteinase K treatment does not cause cleavage of
linker DNA (13). Endogenous peroxidase activity was inhibited by
exposing cells to 3% H2O2 in
phosphate-buffered saline. Cells were equilibrated in a transferase buffer for 5-10 min and then incubated in a reaction mixture
containing biotin-labeled deoxynucleotides and the Klenow fragment of
DNA polymerase at 37 °C. After 60 min, the reaction was stopped, and the biotinylated nucleotides were attached to streptavidin peroxidase. These labeled nucleotides were then detected using an antidigoxigenin antibody conjugated to horseradish peroxidase. To aid detection, the
cells were not counterstained.
Caspase-3 is a downstream effector of the apoptotic response in
osteoblasts. To confirm that the ion pair induced osteoblast apoptosis,
we evaluated the activity of the enzyme in treated cells using a
fluorescent caspase substrate, PhiPhiLuxG1D2 (OncoImmunin Inc.,
Gaithersburg, MD). This reagent becomes fluorescent after interaction
with the activated enzyme, and the increase in fluorescence is
proportional to the change in caspase-3 activity (16). Cells were
treated with the ion pair for time periods ranging from 30 to 275 min.
At each time point, the wells were washed twice and incubated with 10 µM PhiPhiLux-G1D2 for 1 h at 37 °C. Excess
substrate was removed, the cells were washed twice, and the cellular
fluorescence was captured by the confocal microscope.
Apoptosis is dependent on activation of a number of effector enzymes;
inhibition of these enzymes blocks apoptosis. To confirm that the ion
pair induces apoptosis, we inhibited the upstream effector enzyme,
caspase-3, and downstream endonucleases. To block caspase activity,
osteoblasts were incubated with DEVD-CHO (50-300 µM)
(Calbiochem), a specific caspase-3 inhibitor, for 2 h before treatment with 5 mM Pi and 2.9 mM
Ca2+. After 24 h, cell death was evaluated by the MTT
assay. To block endonuclease activity, aurintricarboxylic acid (ATA)
(Sigma) was utilized (17). Cells were incubated with 10-100
µM ATA for 2 h and then treated with 5 mM Pi and 2.9 mM Ca2+.
Again, cell death was evaluated by the MTT assay.
Calcium Channel Blockers and Osteoblast
Apoptosis--
Osteoblasts were treated with a number of different
Ca2+ channel blockers to determine if it was necessary for
[Ca2+]e to enter the cell to activate apoptosis.
Initially, cells were incubated for 2 h with the specific
L-type Ca2+-channel inhibitors, nifedipine
(10-100 µM), and verapamil (10-100 µM)
(18). Then the osteoblasts were treated with 5 mM
Pi and 2.9 mM Ca2+ for a further
24 h. The experiment was repeated using the generalized Ca2+ channel inhibitor, lanthanum chloride (19), and a
specific stretch-activated Ca2+ channel inhibitor,
gadolinium chloride (20). In all cases, cell death was determined using
the MTT procedure.
Measurement of the Mitochondrial Membrane Potential and
Intracellular Ca2+ Distribution--
A combination of
fluorescent probes was used to examine the impact of exogenous
inorganic ions on the cytosolic Ca2+ status and
mitochondrial membrane potential (as indicated in Lemasters et
al. (21)). Cells plated onto 12-well plates were treated with 5 mM Pi and 2.9 mM Ca2+
for 2, 4, and 6 h, as described earlier. At the end of each of these time periods, the medium was replaced with phenol red-free DMEM
containing 1 µM Mitotracker Red (Molecular Probes,
Eugene, OR) or 5 µM Calcium Green 1- AM (Molecular
Probes) for 20-40 min. This medium was then removed and replaced with
fresh phenol red-free DMEM. Cells were then analyzed with the Olympus
Fluoview inverted confocal microscope (Olympus, Melville, NY) with a
long-working distance lens; a specialized cap was used to permit
evaluation of the cells through the plastic dish. To permit
quantification, the plane of maximum fluorescence was determined, and
the photomultiplier tube voltage was set at that point for the control
wells. These parameters were then utilized to measure the relative
fluorescence of the treated cells. Cellular brightness was quantified
using the Fluoview software. For each field of view, the brightness of
20 cells was averaged.
Quantitative Analysis--
Data from the MTT assay were
normalized to control well values and expressed as a percentage of the
control. The values were analyzed using a one-way analysis of variance
test, testing the effect of treatment of each well on cell vitality.
When required, the data were normalized by taking the square roots of
the measured value (22). When that correction did not achieve
normality, a Kruskall-Wallis analysis of variance on ranks was run.
Each cohort of cells removed from the explant seed cultures was treated as a separate replicate. The figures show data representative of
experiments repeated 3-5 times. Significance was assessed when p < 0.05.
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RESULTS |
Ca2+ Activation of Pi-mediated Cell
Death--
When treated with Ca2+ and Pi,
osteoblast-like cells exhibit a dose-dependent decrease in
viability (Fig. 1). It should be noted that in the presence of 1 mM Pi, an elevation
in the extracellular Ca2+ concentration
([Ca2+]e) does not induce cell death. Even if
[Ca2+]e is raised to 9.8 mM, when the
medium Pi concentration is held constant at 1 mM, there is no loss of viability (Fig. 1A).
However, when the medium Pi level is set at 3 mM, a small rise in [Ca2+]e causes a
marked increase in cell death (Fig. 1B). At this
Pi concentration, 2.9 mM
[Ca2+]e decreases osteoblast-like cell viability
by 80%. At higher concentrations of Pi (5 and 7 mM), elevated [Ca2+]e levels increase
the sensitivity of the cells to Pi (Figs. 1, C
and D). At these concentrations, 2.4 mM
[Ca2+]e kills more than 80 and 90% of
osteoblasts, respectively. Fig. 1, A-E, also indicates that
the response of MC-3T3-E1 cells to the ion pair is similar but not
identical to that of osteoblast-like cells. Thus, in comparison with
osteoblast-like cells, MC-3T3-E1 cells show a greater sensitivity to
both 3 mM Pi and 2.4 mM
Ca2+ and 5 mM Pi and 1.9 mM Ca2+. In summary, the combined data set
shown in Fig. 1 indicates that a modest elevation in
[Ca2+]e promotes Pi-mediated cell
death in primary osteoblasts and in MC-3T3-E1 cells.

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Fig. 1.
Effect of Pi and Ca2+
on osteoblast-like cells and MC-3T3-E1 cells. Cells were treated
for 24 h with Ca2+ (1.8-2.9 mM) and
Pi (1-7 mM). Cell viability was assessed with
the MTT assay. A, cells cultured with 1 mM
Pi and increasing concentrations of extracellular calcium
([Ca2+]e). Note that at the highest
[Ca2+]e (10 mM) there was no effect
on MTT activity. B, cells cultured with 3 mM
Pi and increasing concentrations of
[Ca2+]e. At this concentration of Pi,
a small increase in Ca2+ (from 1.9 to 2.9 mM)
induced almost complete killing. C and D, cells
cultured with 5 and 7 mM Pi, respectively, and
varying concentrations of [Ca2+]e. Note that as
the [Ca2+]e is raised, the osteoblasts become
progressively more sensitive to the Pi concentration. The
first series of bars in A-D show control values
(1 mM Pi and 1.8 mM
[Ca2+]e). Values are means and S.E. of the mean
(n = 3). *, p < 0.05, when compared
with control ion concentrations.
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The Ca2+ activation of Pi-mediated cell death
is quite rapid (Fig. 2). By 2 h,
there is a 50% loss of cell viability and by 6 h, more than 80%
of the osteoblasts are dead. A rapid loss of viability is also observed
when MC-3T3-E1 cells are treated with medium containing similar
concentrations of [Ca2+]e and [Pi].
These results together with those shown in Fig. 1 confirm that
[Ca2+]e promotes a sustained elevation in the
rate of induction of death processes in Pi-treated
osteoblasts.

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Fig. 2.
Time course of effect of Pi and
Ca2+ on osteoblast-like cells and MC-3T3-E1 cells.
Cells were treated with 5 mM Pi and 2.9 mM Ca2+ for 2-24 h. Cell viability was
assessed with the MTT assay. Note, within the first 2 h, there was
a significant increase in cell death. By 6 h, more than 80% of
osteoblasts were dead. Each bar represents the mean and S.E.
of the mean (n = 3). *, p < 0.05, when
compared with control.
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Results of three different series of experiments indicate that the
osteoblast dies by apoptosis after treatment with Ca2+ and
Pi. TUNEL analysis suggests that cells treated with 3 mM Pi and 2.9 mM Ca2+
exhibit fragmented DNA. Fig. 3 shows that
the osteoblast-like cells have contracted away from the underlying
matrix and contain TUNEL-positive nuclei. These morphological changes
are characteristic of cells undergoing apoptosis. Use of the
fluorescent caspase-3 substrate, PhiPhiLuxG1D2, indicates that in the
presence of the ion pair, there is a progressive rise in caspase-3
activity (Fig. 4). With time, there is an
elevation in fluorescent cells. Thus, by 30 min, a few faintly positive
cells are evident; by 140 min, many of the cells are positive, and some
cells exhibit a high level of fluorescence. This proportion of positive
cells remains constant through successive time points. It is likely
that the cell to cell variation in fluorescence is due to the
transitory nature of caspase-3 activity. A parallel experiment was
performed to inhibit activities of downstream death effector
endonucleases. When the ion pair concentration is elevated to 5 mM Pi and 2.9 mM Ca2+,
ATA blocks cell death in a dose-dependent manner (Fig.
5A). Likewise, the inhibition
of caspase-3 activity blocks apoptosis (Fig. 5B). At
concentrations above 200 µM, DEVD-CHO, the specific inhibitor of caspase-3, stops cell killing. In summary, a combination of [Ca2+]e and Pi induce cell death,
which is caspase- and endonuclease-dependent and
characterized by the appearance of fragmented DNA. These results are
all consistent with the notion that the ion pair induces osteoblast apoptosis.

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Fig. 3.
TUNEL analysis of osteoblast-like cells
treated with Pi and Ca2+. Cells in 24-well
culture plates were treated with 3 mM Pi and
2.9 mM Ca2+ for 24 h. TUNEL analysis was
performed on both treated (B) and control (A)
cells. Note that the untreated osteoblasts are flattened, indistinct,
and TUNEL-negative. After treatment with the apoptogen, the cells
are raised (white triangles), and the nuclei are
TUNEL-positive (black triangles). Magnification,
×200.
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Fig. 4.
Activation of caspase-3 by treatment with
Pi and Ca2+. Osteoblasts were treated with
5 mM Pi and 2.9 mM Ca2+
for the indicated time periods. Cells were then treated with the
fluorescent caspase-3 substrate, 10 µM PhiPhiLux-G2 for
1 h at 37 °C. Images of the cells were then captured using the
Olympus Fluoview confocal microscope. Note the progressive increase in
cellular fluorescence, indicating activation of caspase-3, a critical
apoptosis effector enzyme. Magnificaton, ×400.
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Fig. 5.
Effect of apoptosis inhibitors on
Pi and Ca2+- mediated osteoblast death.
Osteoblasts were pretreated with ATA (A) or DEVD-CHO
(B) for 2 h. Cells were then cultured for 24 h in
the presence of 5 mM Pi and 2.9 mM
Ca2+. Cell viability was assessed by the MTT assay. At
concentrations above 40 µM, ATA, an endogenous
endonuclease inhibitor, completely blocked osteoblast death. Likewise,
DEVD-CHO, an inhibitor of caspase-3, at concentrations above 100 µM prevented cell killing. Both of these enzymes are
downstream effectors of the apoptotic process. Each bar
represents the mean and S.E. of the mean (n = 3). *,
p < 0.05, when compared with control Ca2+
and Pi concentrations; #, p < 0.05, when compared with cells treated with 5 mM Pi
and 2.9 mM Ca2+.
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Inhibition of Ca2+ and Pi Transport on
Osteoblast Death--
When Pi transport is blocked, the
ion pair fails to trigger cell death (Fig.
6). Thus, over 80% of the osteoblasts
die when exposed to 2.9 mM Ca2+ and 3 mM Pi. The presence of PFA, an inhibitor of
Pi transport, completely abrogates osteoblast apoptosis
(Fig. 6A). Indeed, even if the Pi concentration
is raised to 7 mM, PFA blocks cell death. PFA also protects
MC-3T3-E1 cells from apoptosis. Fig. 6B shows that if the
medium contains PFA, the cells retain their viability even when treated
with high levels of the ion pair (2.9 Ca2+ and 7 mM Pi). PFA alone has no effect on bone cell
MTT activity. Results of this series of experiments suggest that uptake
or binding of Pi by the treated cells is a requirement of
the apoptotic process.

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Fig. 6.
Effect of PFA on Pi- and
Ca2+-mediated apoptosis. Osteoblast-like cells
(A) and MC-3T3-E1 cells (B) were cultured for
24 h with 2.9 mM Ca2+ and 3-7
mM Pi in the presence and absence of 5 mM PFA. Cell viability was assessed by the MTT assay. Note
that treatment with PFA blocked Ca2+- and
Pi-induced death of both osteoblast-like cells and
MC-3T3-E1 cells. The first bar in A and
B shows values for cells treated with 1 mM
Pi and 1.9 mM Ca2+
(Control). Each bar represents the mean and S.E.
of the mean (n = 3). *, p < 0.05, when
compared with controls.
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Although Pi transporters are required for
Ca2+-Pi-induced cell death, inhibitors of
Ca2+ channel transport exert little effect on ion
pair-mediated osteoblast apoptosis. Treatment with verapamil or
nifedipine effected no significant change in
Ca2+-dependent Pi-mediated bone
cell death. Table I shows that at all
concentrations evaluated, these specific L-type
Ca2+ channel blockers fail to increase the percentage of
viable cells. Lanthanum chloride, a general Ca2+ channel
inhibitor, appears to offer some protection from ion pair-induced
apoptosis. At a concentration of 100 µM, this agent doubles the number of vital cells. However, since high lanthanum chloride levels are toxic to cells, it is probable that the increase in
vitality is due to the lanthanides precipitating apatite out of
solution (23). Deposition of the mineral phase would lower the
activity of the ion pair as well as reducing the concentration of
lanthanum chloride to nontoxic levels. Gadolinium chloride, an
inhibitor of the stretch-activated (SA-Cat) Ca2+ channels
also provides no protection from Ca2+- and
Pi-induced apoptosis (data not shown). Together, results of
these experiments indicate that inhibition of Ca2+
transport through a number of common channels has a minimal effect in
blocking Ca2+ dependent Pi-mediated bone cell
apoptosis.
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Table I
Effect of calcium channel blockers on calcium and phosphate-mediated
osteoblast apoptosis
Each of the experimental groups was treated with 5 mM
Pi and 2.9 mM Ca2+ along with the indicated
concentration of Ca2+ channel blocker for 24 h. The number
of viable cells was determined using the MTT assay. Verapamil and
nifedipine failed to protect the cells from apoptosis. Lanthanum
chloride, a general Ca2+ channel inhibitor, provided some
protection but only at high (100 µM) concentrations.
Values shown are the mean and S.E. (n = 3).
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Mechanism of Ca2+- and Pi-mediated Cell
Death--
Confocal microscopy indicates that mitochondria are
involved in the activation of apoptosis (Figs.
7 and 8).
When probed with Mitotracker Red (500 nM), a membrane
voltage dye, osteoblasts display a low level of mitochondrial
fluorescence (Fig. 7A). Two h after treatment with 5 mM Pi and 2.9 mM Ca2+
there is a transient but marked increase in Mitotracker fluorescence (Fig. 7B); by 4 h, the fluorescence has returned to
base-line values (Fig. 7C). Six hours after treatment, the
cells are shrunken, cell to cell contacts are lost, and many of the
cells are dead (Fig. 7D). The residual fluorescence of these
cells is probably due to the dye binding to membrane remnants. The
change in mitochondrial fluorescence is shown graphically in Fig.
9A. Treatment with the apoptogen for 2 h elicited a
3-fold increase in fluorescence. Mitochondrial fluorescence returns to
base-line values by 4 h; the figure also shows that Mitotracker
binds to the cell remnants, evidencing a small but significant increase
in fluorescence at 6 h. Calcium fluorescence, on the other hand,
is extremely low in the control cells (Fig. 8A). In contrast
to mitochondrial fluorescence, where a spike in fluorescence is seen
after 2 h but then returns to base-line values, intracellular
Ca2+ increases slowly, and a peak is reached 4 h after
treatment (Fig. 8, B and C). By 6 h, when
apoptosis in complete, intracellular calcium fluorescence is below
base-line values (Fig. 8D). The change in Ca2+
fluorescence is show graphically in Fig.
9B. A
time-dependent increase in calcium-green fluorescence is
evident, and peak values are seen 4 h after treatment. When values
shown in Fig. 9, A and B, are compared it is
evident that peak Ca2+ levels are reached after loss of
mitochondrial fluorescence.

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Fig. 7.
Fluorescence of osteoblast-like cells treated
with 5 mM Pi and 2.9 mM
Ca2+ and probed with Mitotracker Red. Cells were
treated with the ion pair and then probed with Mitotracker Red (500 nM). The fluorescent probe was added to each sample, and
the change in fluorescence was recorded over a 6-h period with an
Olympus Fluoview confocal microscope. Throughout the experiment, the
photomultiplier voltages were held constant to allow quantification.
A, control. B, 2-h treatment. C, 4-h
treatment. D, 6-h treatment. Note the increase in
mitochondrial membrane fluorescence at 2 h. By 6 h, the cells
were shrunken, and most were dead. The residual fluorescence was
probably due to the dye binding to cell fragments. Magnification,
×600.
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Fig. 8.
Fluorescence of osteoblast-like cells treated
with 5 mM Pi and 2.9 mM
Ca2+ and probed with Calcium Green-AM. Cells were
treated with Pi and Ca2+ and then incubated
with Calcium Green 1-AM (5 µM). The change in
fluorescence over a 6-h period was recorded by confocal microscopy.
Throughout the experiment, photomultiplier voltages were held constant
to allow quantification. A, control. B, 2-h
treatment. C, 4-h treatment. D, 6-h treatment.
Note the initial low level of cytosolic Ca2+ fluorescence
in these cells. However, by 4 h a significant increase in
fluorescence was apparent. Magnification, ×600.
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Fig. 9.
Quantification of mitochondrial membrane and
intracellular Ca2+ fluorescence. The fluorescence
yield of cells imaged in Figs. 7 and 8 were measured with the Fluoview
software. 20 cells from each field were defined digitally, and the
brightness was quantified and averaged. A, mitochondrial
fluorescence. B, intracellular Ca2+
fluorescence. Note that the peak values of time-dependent
Calcium Green-AM fluorescence, an indicator of the cytoplasmic
Ca2+ concentration, was maximal after loss of Mitotracker
Red fluorescence. Since Mitotracker Red values provided an index of the
mitochondrial membrane potential, it is probable that loss of
mitochondrial function precedes other cytoplasmic events. Each
bar represents the mean and S.E. of the mean
(n = 20). *, p < 0.05, when compared
with control time point. #, p < 0.05, when compared
with the 2-h time point.
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DISCUSSION |
Using two different bone cell preparations (primary human
osteoblasts and MC-3T3-E1 cells), we demonstrated that Ca2+
enhanced Pi-dependent apoptosis and that the
impact of the ion pair was far more profound that exhibited by
Pi alone (1). The effects of the individual ions on cell
viability provided clues to the mechanism by which these apatite
components activate the death program. Whereas increased
[Ca2+]e alone had no effect on osteoblast
apoptosis, the combined ion pair was a powerful inducer of the death
response (Fig. 1). When Pi transport was inhibited, there
was almost complete protection from
[Ca2+]e-Pi-mediated apoptosis (Fig.
6). Moreover, since a number of specific Ca2+ channel
blockers failed to inhibit apoptosis (Table I), the results suggest
that Ca2+ may interact with Pi at the level of
the cell membrane and that this event mediates intracellular
Pi-induced osteoblast death. In terms of the in
vivo relevance of these findings, it is probable that at sites of
bone resorption, there is a localized increase in both ions. Indeed
Silver et al. (24) show that in close proximity to
osteoclasts, extracellular Ca2+ levels can be elevated
40-fold. On this basis, we suggest that, at resorption locales, a
limited rise in the local ion pair concentration would activate
apoptosis in osteoblasts as well as other vicinal cells. Accordingly,
this finding would explain why many of the osteoblasts present at
bone-remodeling sites undergo apoptotic death (4).
Results of the investigation confirm and extend earlier observations
that low levels of Pi caused chondrocyte and osteoblast apoptosis (1, 13). The current investigation that was focused on
Ca2+ indicated that when the concentration of this ion was
raised from 1.8 to 2.9 mM, there was a dramatic increase in
Pi-mediated osteoblast death (Fig. 1). It should be noted
that the rise in [Ca2+]e was modest. The highest
Ca2+ concentration used (2.9 mM) was less than
twice the level present in normal serum and much lower than the values
reported at bone-remodeling sites (24). That death was through
apoptosis was confirmed utilizing the TUNEL assay (Fig. 3), the
microscopic appearance of the cells, caspase measurements (Fig. 4), and
inhibitor studies (Fig. 5). Thus, in the presence of the ion pair, the
cells exhibited characteristics of apoptosis, including contraction of
the cells, release of osteoblasts from the substratum and loss of
contact with neighboring cells as well as marked change in the
mitochondrial membrane potential. In addition, when the activity of
both caspase-3 and endogenous endonucleases was locked, there was
inhibition of apoptosis. Since activation of these enzymes is a
hallmark of apoptosis, it is evident that an elevation in the local
Ca2+ and Pi concentration serves as a potent
osteoblastic apoptogen.
We explored the mechanism by which Ca2+ activates
Pi-mediated cell death by assuming that Ca2+
synergizes anion entry into osteoblasts. This viewpoint was supported by the observation that at low Pi concentrations, an
elevation in [Ca2+ ]e did not activate cell
death. Thus, possible toxic effects of this cation on osteoblast
function could be ignored. When the medium Pi content was
elevated, a rise in [Ca2+]e caused a rapid (6 h)
induction of killing (Fig. 2). The importance of Pi was
further emphasized by the finding that PFA, an agent that blocks
Na-Pi co-transporters and prevents Pi entry
into cells, inhibited ion pair-dependent osteoblast death (Fig. 6). As for a direct role for Ca2+ in osteoblast
apoptosis, we explored the possibility that an increase in
[Ca2+]e could elevate the intracellular
Ca2+ concentration and thereby promote the death cascade.
However, when osteoblasts were treated with a range of Ca2+
channel blockers, ion pair-induced apoptosis was not inhibited (Table
I). Thus, although cell death was dependent on the presence of both
ions, apoptosis was not activated by a flood of
[Ca2+]e into the treated cells.
The channel blocker experiments discussed above indicated that bone
cell apoptosis was mediated by more subtle processes, possibly related
to a change in mitochondrial function. Studies of other apoptotic
systems indicate that mitochondrial dysfunction results in release of
Ca2+ from intracellular stores; a rise in intracellular
Ca2+ and loss of cytochrome c serve to activate
downstream events in the apoptotic process (25, 26). In bone cells,
when the mitochondrial dye, Mitotracker Red, was used to follow the
response of mitochondria to the ion pair, an initial hyperpolarization of the mitochondrial signal was seen (Fig. 7B). However,
4 h after treatment, the signal had dropped below that of the
control cells (Fig. 7C). In the same time period, there was
an elevation in intracellular Ca2+ fluorescence (Fig.
8C). By 6 h, the cells showed clear morphological evidence of death; both mitochondria and intracellular Ca2+
signals had greatly decreased (Figs. 7D and 8D).
Thus, the initial hyperpolarization of the mitochondria was an
immediate reaction to the apoptogens. Subsequently, there was a change
in Ca2+ flux in the treated cells. It is tempting to
speculate that the increase in Ca2+ fluorescence was
directly related to the loss of mitochondrial membrane potential and
the subsequent inability of the mitochondria and calciosome to maintain
intracellular Ca2+ levels. When this stage is reached, the
transient rise in the cytosolic Ca2+ concentration may have
served to activate downstream effectors of the apoptotic pathway.
Aside from the mechanisms discussed above, there are a number of other
ways in which [Ca2+]e could synergize
Pi-mediated osteoblast apoptosis. [Ca2+]e and Pi may complex at the
cell membrane and induce death as an ion pair, ion cluster, or
particle. The complexed ions could bind to sites on the membrane and
trigger receptor-mediated transduction pathways that affect the
apoptotic response. Alternatively, if particles are formed at the
plasma membrane, they could be endocytosed into the cell and trigger
apoptosis directly. In favor of particle formation is the finding that
variously sized calcium phosphate particles can undergo endocytosis
(27-29). Indeed, Kingsmill and Boyde (30) point out that in mandibular
bone, dead osteocytes contain mineral particles. Pertinent to this
observation, two recent reports indicate that if the particle size is
small, then crystals of hydroxyapatite can induce osteoblast apoptosis
(31, 32). On the other hand, the observation that PFA blocks osteoblast apoptosis lends strong support to the view that to induce apoptosis, Pi needs to enter the cell through a specific plasma
membrane Na-Pi transporter. Irrespective of the mechanism,
the results support our original contention that two major products of
the resorptive process serve as a powerful local apoptogen.