Matrix Regulation of Skeletal Cell Apoptosis

ROLE OF CALCIUM AND PHOSPHATE IONS*

Christopher S. AdamsDagger, Kyle Mansfield, Robert L. Perlot, and Irving M. Shapiro

From the Department of Biochemistry, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6002

Received for publication, July 20, 2000, and in revised form, February 23, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previously, we noted that inorganic phosphate (Pi), a major component of bone extracellular matrix, induced osteoblast apoptosis (Meleti, Z., Shapiro, I. M., and Adams, C. S. (2000) Bone (NY) 27, 359-366). Since Ca2+ along with Pi is released from bone during the resorption process, we advanced the hypothesis that Ca2+ modulates Pi-mediated osteoblast apoptosis. To test this hypothesis, osteoblasts were incubated with both ions, and cell death was determined. We noted that a modest increase in the medium Ca2+ concentrations ([Ca2+]e) of 0.1-1 mM caused a profound and rapid enhancement in Pi-dependent death of cultured osteoblasts. An elevation in [Ca2+]e alone had no effect on osteoblast viability, whereas Ca2+ channel blockers failed to inhibit killing of ion pair-treated cells. These results indicated that Pi-mediated cell death is not dependent on a sustained increase in the cytosolic Ca2+ concentration. Terminal dUTP nick-end labeling analysis and measurement of caspase-3 activity of the ion pair-treated cells suggested that death was apoptotic. Apoptosis was confirmed using caspase-3 and endonuclease inhibitors. The mitochondrial membrane potential and cytosolic Ca2+ status of the treated cells were evaluated. After incubation with [Ca2+ ]e and Pi, a decrease in mitochondrial fluorescence was noted, suggesting that the ions decreased the mitochondrial transmembrane potential. Subsequent to the fall in mitochondrial membrane potential, there was a transient elevation in the cytosolic Ca2+ concentration. Results of the study suggest that the ion pair conspire at the level of the plasma membrane to induce intracellular changes that result in loss of mitochondrial function. The subsequent increase in the cytosolic Ca2+ concentration may trigger downstream events that transduce osteoblast apoptosis.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 beta -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.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

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.

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+.

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.

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).

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.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

    FOOTNOTES

* This work was supported by National Institutes of Health Grants DE-13319, DE-10875, and DE-AR05748.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.

Dagger To whom correspondence should be addressed: Dept. of Biochemistry, School of Dental Medicine, University of Pennsylvania, 4010 Locust St., Philadelphia, PA 19104-6002. Tel.: 215-898-9167; Fax: 215-898-3695; Adamsc@biochem.dental.upenn.edu

Published, JBC Papers in Press, March 14, 2001, DOI 10.1074/jbc.M006492200

    ABBREVIATIONS

The abbreviations used are: DMEM, Dulbecco's modified eagle's medium; PFA, phosphonoformic acid; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; TUNEL, terminal dUTP nick-end labeling; DEVD- CHO, Asp-Glu-Val-Asp-aldehyde; ATA, aurintricarboxylic acid.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.