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• W2089947504 abstract "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. 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. Dulbecco's modified eagle's medium phosphonoformic acid 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide terminal dUTP nick-end labeling Asp-Glu-Val-Asp-aldehyde aurintricarboxylic acid Bone adapts to mechanical and physiological stress by a unique form of tissue replacement contained within discrete structures defined as basic multicellular units (2Frost H.M. N. Eng. J. Med. 1973; 289: 864-865PubMed Google Scholar). 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 (3Bronckers A.L.J.J. Goei W. Luo G. Karsenty G. D'Souza R.N. Lyaruu D.M. Burger E.H. J. Bone Miner. Res. 1996; 11: 1281-1291Crossref PubMed Scopus (136) Google Scholar). Thus, there is evidence of DNA fragmentation in osteoclasts, osteoblasts, and osteocytes (4Hughes D.E. Dai A. Tiffee J.C. Li H.H. Mundy G.R. Boyce B.F. Nat. Med. 1996; 2: 1132-1136Crossref PubMed Scopus (705) Google Scholar, 5Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (472) Google Scholar). 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 (5Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (472) Google Scholar, 6Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1999; 104: 439-446Crossref PubMed Scopus (896) Google Scholar, 7Noble B.S. Stevens H. Loveridge N. Reeve J. Bone. 1997; 20: 273-282Crossref PubMed Scopus (199) Google Scholar, 8Weinstein R.S. Jilka R.L. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1998; 102: 274-282Crossref PubMed Scopus (1428) Google Scholar). 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 (9Takahashi N. Akatsu T. Udagawa N. Sasaki T. Yamaguchi A. Moseley J.M. Martin T.J. Suda T.,. Endocrinology. 1988; 123: 2600-2602Crossref PubMed Scopus (856) Google Scholar). 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 (5Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (472) Google Scholar, 6Jilka R.L. Weinstein R.S. Bellido T. Roberson P. Parfitt A.M. Manolagas S.C. J. Clin. Invest. 1999; 104: 439-446Crossref PubMed Scopus (896) Google Scholar, 10Hill P.A. Tumber A. Meikle M.C. Endocrinology. 1997; 138: 3849-3858Crossref PubMed Scopus (186) Google Scholar). In addition, it has been demonstrated that a number of pharmacological agents can induce osteoblast apoptosis in vitro (5Jilka R.L. Weinstein R.S. Bellido T. Parfitt A.M. Manolagas S.C. J. Bone Miner. Res. 1998; 13: 793-802Crossref PubMed Scopus (472) Google Scholar, 10Hill P.A. Tumber A. Meikle M.C. Endocrinology. 1997; 138: 3849-3858Crossref PubMed Scopus (186) Google Scholar, 11Kitajima I. Nakajima T. Imamura T. Takasaki I. Kawahara K. Okano T. Tokioka T. Soejima Y. Abeyama K. Maruyama I. J. Bone Miner. Res. 1996; 11: 200-210Crossref PubMed Scopus (64) Google Scholar, 12Ihbe A. Baumann G. Heinzmann U. Atkinson M.J. Calcif. Tissue Int. 1998; 63: 208-213Crossref PubMed Scopus (11) Google Scholar). 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 (1Meleti Z. Shapiro I.M. Adams C.S. Bone ( NY ). 2000; 27: 359-366Crossref PubMed Scopus (210) Google Scholar, 13Mansfield K. Rajpurohit R. Shapiro I.M. J. Cell. Physiol. 1999; 179: 276-286Crossref PubMed Scopus (100) Google Scholar). 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. 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 mml-glutamine, and antibiotics. The Piconcentration 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 mml-glutamine, 5 mm β-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 Meletiet al. (1Meleti Z. Shapiro I.M. Adams C.S. Bone ( NY ). 2000; 27: 359-366Crossref PubMed Scopus (210) Google Scholar)). 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 (14Franceschi R.T. Iyer B.S. J. Bone Miner. Res. 1992; 7: 235-246Crossref PubMed Scopus (480) Google Scholar). These cells were grown to confluence in DMEM supplemented with 10% fetal bovine serum, 2 mml-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). Osteoblast viability was assessed as a function of the Pi concentration and [Ca2+]e as well as treatment time. Piwas 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 mmCa2+, 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. 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 (15McGahon A.J. Martin S.J. Bissonnette R.P. Mahboubi A. Shi Y. Mogil R.J. Nishioka W.K. Green D.R. Methods Cell Biol. 1995; 46: 153-185Crossref PubMed Scopus (523) Google Scholar). 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. Although we have previously demonstrated that Pi induced osteoblast apoptosis (1Meleti Z. Shapiro I.M. Adams C.S. Bone ( NY ). 2000; 27: 359-366Crossref PubMed Scopus (210) Google Scholar), 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 (13Mansfield K. Rajpurohit R. Shapiro I.M. J. Cell. Physiol. 1999; 179: 276-286Crossref PubMed Scopus (100) Google Scholar). 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 (16Komoriya A. Packard B.Z. Brown M.J. Wu M.L. Henkart P.A. J. Exp. Med. 2000; 191: 1819-1828Crossref PubMed Scopus (138) Google Scholar). 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 mmCa2+. After 24 h, cell death was evaluated by the MTT assay. To block endonuclease activity, aurintricarboxylic acid (ATA) (Sigma) was utilized (17Andrew D.J. Hay A.M. Evans S.W. Immunopharmacology. 1999; 41: 1-10Crossref PubMed Scopus (22) Google Scholar). 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. 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 specificl-type Ca2+-channel inhibitors, nifedipine (10–100 μm), and verapamil (10–100 μm) (18Atlas D. Adler M. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 1237-1241Crossref PubMed Scopus (101) Google Scholar). Then the osteoblasts were treated with 5 mmPi and 2.9 mm Ca2+ for a further 24 h. The experiment was repeated using the generalized Ca2+ channel inhibitor, lanthanum chloride (19Radding W. Jordan S.E. Hester R.B. Blair H.C. Exp. Cell Res. 1999; 253: 689-696Crossref PubMed Scopus (20) Google Scholar), and a specific stretch-activated Ca2+ channel inhibitor, gadolinium chloride (20Ryder K.D. Duncan R.L. Calcif. Tissue Int. 2000; 67: 241-246Crossref PubMed Scopus (52) Google Scholar). In all cases, cell death was determined using the MTT procedure. 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. (21Lemasters J.J. Nieminen A.L. Qian T. Trost L.C. Elmore S.P. Nishimura Y.X.C.R. Cascio W.E. Bradham C.A. Brenner D.A. Herman B. Biochim. Biophys. Acta. 1998; 1366: 177-196Crossref PubMed Scopus (1230) Google Scholar)). 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. 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 (22Snedecor G.W. Cochran W.G. Statistical Methods. 6th Ed. The Iowa State University Press, Ames, Iowa1967Google Scholar). 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 whenp < 0.05. 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. 1 A). 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. 1 B). 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, Cand 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 mmCa2+ 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. 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. 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.5 A). Likewise, the inhibition of caspase-3 activity blocks apoptosis (Fig. 5 B). 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.Figure 4Activation 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 5Effect 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 mmCa2+. 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 barrepresents 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 Piand 2.9 mm Ca2+.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 6 A). 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. 6 B 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. 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 (23Mayer I. Layani J.D. Givan A. Gaft M. Blanc P. J. Inorg. Biochem. 1999; 73: 221-226Crossref PubMed Scopus (51) Google Scholar). 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.Table IEffect of calcium channel blockers on calcium and phosphate-mediated osteoblast apoptosisControl0 μm25 μm50 μm100 μmVerapamil100.1 (1.62)28.8 (5.98) 1-ap < 0.05 from control values.16.1 (2.33) 1-ap < 0.05 from control values.15.9 (3.76) 1-ap < 0.05 from control values.23.8 (2.20) 1-ap < 0.05 from control values.Nifedipine100.1 (1.62)33.8 (5.98) 1-ap < 0.05 from control values.31.8 (1.56) 1-ap < 0.05 from control values.33.8 (2.51) 1-ap < 0.05 from control values.41.2 (3.35) 1-ap < 0.05 from control values.Lanthanum chloride100.1 (1.62)20.1 (2.55) 1-ap < 0.05 from control values.24.4 (0.93) 1-ap < 0.05 from control values.22.5 (2.62) 1-ap < 0.05 from control values.57.0 (5.40) 1-bp < 0.05 from 0 μm values.Each of the experimental groups was treated with 5 mmPi 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).1-a p < 0.05 from control values.1-b p < 0.05 from 0 μm values. Open table in a new tab Each of the experimental groups was treated with 5 mmPi 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). 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. 7 A). Two h after treatment with 5 mm Pi and 2.9 mm Ca2+there is a transient but marked increase in Mitotracker fluorescence (Fig. 7 B); by 4 h, the fluorescence has returned to base-line values (Fig. 7 C). Six hours after treatment, the cells are shrunken, cell to cell contacts are lost, and many of the cells are dead (Fig. 7 D). 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.9 A. 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" @default.
• W2089947504 created "2016-06-24" @default.
• W2089947504 creator A5019521528 @default.
• W2089947504 creator A5034557839 @default.
• W2089947504 creator A5059642352 @default.
• W2089947504 creator A5087422768 @default.
• W2089947504 date "2001-01-01" @default.
• W2089947504 modified "2023-10-14" @default.
• W2089947504 title "Matrix Regulation of Skeletal Cell Apoptosis" @default.
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