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Induction of Apoptosis in Skeletal Tissues: Phosphate-Mediated Chick Chondrocyte Apoptosis is Calcium Dependent

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Abstract

In an earlier study, we have shown that Pi induced apoptosis of terminally differentiated hypertrophic chondrocytes. To ascertain whether Ca2+ modulates Pi-induced cell death, we asked the following two questions: First, can we prevent Pi-induced apoptosis by removing Ca2+ from the culture medium; alternatively, can we potentiate cell death by increasing the Ca2+ concentration? Second, can we inhibit chondrocyte apoptosis by blocking Pi transport? We also explored the mechanism of apoptosis by evaluating mitochondrial activity and reactive oxygen species (ROS) generation in cells treated with the ion pair. We noted that EDTA and EGTA blocked Pi-induced apoptosis in a dose-dependent manner. While high levels of Ca2+ alone had little effect on chondrocyte viability, the cation enhanced Pi-dependent cell death and greatly increased Pi uptake. When Pi transport was blocked, there was complete inhibition of cell killing. The process of cell death was characterized by mitochondrial hyperpolarization; two hours following apoptogen treatment, there was a significant decrease in the mitochondrial membrane potential. Coincident with the changes in mitochondrial function, there was an increase in intracellular Ca2+ that was maintained throughout the experimental period. A raised Ca2+ signal was observed in blebs at the cell membrane. Finally, we noted that, 75 minutes after treatment with the ion pair, there was a six-fold elevation in ROS levels. This increase declined to baseline values after three hours. Based on these observations, we suggest that, at the cartilage mineralization front, an elevation in local environmental Ca2+ and Pi concentrations modulates oxidative metabolism, and triggers apoptosis of terminally differentiated chondrocytes.

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References

  1. ALJJ Bronckers W Goei G Luo G Karsenty RN D’Souza DM Lyaruu EH Burger (1996) ArticleTitleDNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling. J Bone Miner Res 11 1281–1291 Occurrence Handle1:CAS:528:DyaK28Xls1ChtLY%3D Occurrence Handle8864903

    CAS  PubMed  Google Scholar 

  2. CE Farnum NJ Wilsman (1987) ArticleTitleMorphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage. Anat Rec 219 221–232 Occurrence Handle1:STN:280:BieC3cvmtlA%3D Occurrence Handle3425941

    CAS  PubMed  Google Scholar 

  3. CE Farnum NJ Wilsman (1989) ArticleTitleCellular turnover at the chondro–osseous junction of growth plate cartilage: Analysis by serial sections at the light microscopical level. J Orthop Res 7 654–666 Occurrence Handle1:STN:280:BiaA2c7pvFY%3D Occurrence Handle2760737

    CAS  PubMed  Google Scholar 

  4. CE Farnum NJ Wilsman (1989) ArticleTitleCondensation of hypertrophic chondrocytes at the condro–osseous junction of growth plate cartilage in yucatan swine: Relationship to long bone growth. Am J Anat 186 346–358 Occurrence Handle1:STN:280:By%2BD2s7osFQ%3D Occurrence Handle2589219

    CAS  PubMed  Google Scholar 

  5. GJ Gibson WJ Kohler MB Schaffler (1995) ArticleTitleChondrocyte apoptosis in endochondrial ossification of chick sterna. Dev Dyn 203 468–476 Occurrence Handle1:STN:280:BymD3Mnls1Q%3D Occurrence Handle7496038

    CAS  PubMed  Google Scholar 

  6. M Hatori KJ Klatte CC Teixeira IM Shapiro (1995) ArticleTitleEnd labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosisin terminally differentiated chondrocytes. J Bone Miner Res 10 1960–1968 Occurrence Handle1:STN:280:BymC1M3ntFM%3D Occurrence Handle8619377

    CAS  PubMed  Google Scholar 

  7. Y Ishizaki JF Burne MC Raff (1994) ArticleTitleAutocrine signals enable chondrocytes to survive in culture. J Cell Biol 126 1069–1077 Occurrence Handle1:CAS:528:DyaK2cXltVGhtL8%3D Occurrence Handle8051206

    CAS  PubMed  Google Scholar 

  8. Y Ishizaki L Cheng AW Mudge MC Raff (1995) ArticleTitleProgrammed cell death by default in embryonic cells, fibroblasts, and cancer cells. Mol Biol Cell 6 1443–1458 Occurrence Handle1:CAS:528:DyaK2MXpsFejsb4%3D Occurrence Handle8589448

    CAS  PubMed  Google Scholar 

  9. D Mitrovic M Quintero A Stankovic A Ryckewaert (1983) ArticleTitleCell density of adult human femoral condylar articular cartilage. Joints with normal and fibrillated surfaces. Lab Invest 49 309–316 Occurrence Handle1:STN:280:BiyB1MrlvFQ%3D Occurrence Handle6193331

    CAS  PubMed  Google Scholar 

  10. M Quintero D Mitrovic A Stankovic S de Seze L Miravet A Ryckewaert (1984) ArticleTitleAspects Cellulaires du viellissement du cartilage articulaire I. Cartilage condylien a surface normale, preleve dans les genoux normaux. Rev Rhum 51 375–379 Occurrence Handle1:STN:280:BiqD2c%2FgsFU%3D Occurrence Handle6494776

    CAS  PubMed  Google Scholar 

  11. M Quintero D Mitrovic A Stankovic S de Seze L Miravet A Ryckewaert (1984) ArticleTitleAspects cellulaires du viellissement du cartilage articulaire II. Cartilage condylien a surface fissuree preleve dans les genoux “normaux” et “arthrosiques.” Rev Rhum 51 445–449 Occurrence Handle1:STN:280:BiqD28rls1c%3D Occurrence Handle6505594

    CAS  PubMed  Google Scholar 

  12. E Vignon M Arlot LM Patricot G Vignon (1976) ArticleTitleThe cell density of human femoral head cartilage. Clin Orthop 121 303–308 Occurrence Handle991515

    PubMed  Google Scholar 

  13. MC Hochberg M Lethbridge–Cejku WW Scott Jr CC Plato JD Tobin (1993) ArticleTitleObesity and osteoarthritis of the hands in women. Osteoarthritis Cartilage 1 129–135 Occurrence Handle1:STN:280:ByiD2c%2FnvFM%3D Occurrence Handle8886089

    CAS  PubMed  Google Scholar 

  14. M Lethbridge–Cejku JD Tobin WW Scott Jr R Reichle CC Plato MC Hochberg (1994) ArticleTitleThe relationship of age and gender to prevalence and pattern of radiographic changes of osteoarthritis of the knee: data from Caucasian participants in the Baltimore Longitudinal Study of Aging. Aging (Milano) 6 353–357 Occurrence Handle1:STN:280:ByqB3c%2FpvFc%3D

    CAS  Google Scholar 

  15. CS Adams WE Horton (1998) ArticleTitleChondrocyte apoptosis increases with age in the articular cartilage of adult animals. Anat Rec 250 418–425 Occurrence Handle10.1002/(SICI)1097-0185(199804)250:4<418::AID-AR4>3.3.CO;2-B Occurrence Handle1:STN:280:DyaK1c3is1WrsQ%3D%3D Occurrence Handle9566531

    Article  CAS  PubMed  Google Scholar 

  16. K Mansfield R Rajpurohit IM Shapiro (1999) ArticleTitleExtracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes. J Cell Physiol 179 276–286 Occurrence Handle10.1002/(SICI)1097-4652(199906)179:3<276::AID-JCP5>3.0.CO;2-# Occurrence Handle1:CAS:528:DyaK1MXjtVaksL0%3D Occurrence Handle10228946

    Article  CAS  PubMed  Google Scholar 

  17. K Mansfield CC Teixeira CS Adams IM Shapiro (2001) ArticleTitlePhosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism. Bone 28 1–8 Occurrence Handle10.1016/S8756-3282(00)00409-9 Occurrence Handle1:CAS:528:DC%2BD3MXptlWltA%3D%3D Occurrence Handle11165936

    Article  CAS  PubMed  Google Scholar 

  18. R Rajpurohit K Mansfield K Ohyama D Ewert IM Shapiro (1999) ArticleTitleChondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate. J Cell Physiol 179 287–296 Occurrence Handle10.1002/(SICI)1097-4652(199906)179:3<287::AID-JCP6>3.0.CO;2-T Occurrence Handle1:CAS:528:DyaK1MXjtVaksLo%3D Occurrence Handle10228947

    Article  CAS  PubMed  Google Scholar 

  19. CS Adams K Mansfield RL Perlot IM Shapiro (2001) ArticleTitleMatrix regulation of skeletal cell apoptosis—Role of calcium and phosphate ions. J Biol Chem 276 20316–20322 Occurrence Handle10.1074/jbc.M006492200 Occurrence Handle1:CAS:528:DC%2BD3MXktlKntrY%3D Occurrence Handle11278303

    Article  CAS  PubMed  Google Scholar 

  20. R Rajpurohit CJ Koch Z Tao CM Teixeira IM Shapiro (1996) ArticleTitleAdaptation of chondrocytes to low oxygen tension: relationship between hypoxia and cellular metabolism. J Cell Physiol 168 424–432 Occurrence Handle10.1002/(SICI)1097-4652(199608)168:2<424::AID-JCP21>3.0.CO;2-1 Occurrence Handle1:CAS:528:DyaK28XkslCgs7w%3D Occurrence Handle8707878

    Article  CAS  PubMed  Google Scholar 

  21. O Oshima PS Leboy SA McDonald RS Tuan IM Shapiro (1989) ArticleTitleDevelopmental expression of genes in chick growth cartilage detected by in situ hybridization. Calcif Tissue Int 45 182–192 Occurrence Handle1:CAS:528:DyaK3cXjslei Occurrence Handle2505910

    CAS  PubMed  Google Scholar 

  22. AJ McGahon SJ Martin RP Bissonnette A Mahboubi Y Shi RJ Mogil WK Nishioka DR Green (1995) ArticleTitleThe end of the (cell) line: methods for the study of apoptosis in vitro. Meth Cell Biol 46 153–185 Occurrence Handle1:STN:280:ByqA3szptlc%3D

    CAS  Google Scholar 

  23. GD Baxter RJ Collins BV Harmon S Kumar RL Prentice PJ Smith MF Lavin (1989) ArticleTitleCell death by apoptosis in acute leukaemia. J Pathol 158 123–129 Occurrence Handle1:STN:280:BiaA38fms10%3D Occurrence Handle2754542

    CAS  PubMed  Google Scholar 

  24. Z Meleti IM Shapiro CS Adams (2000) ArticleTitleInorganic phosphate induces apoptosis of osteoblast-like cells in culture. Bone 27 359–366 Occurrence Handle10.1016/S8756-3282(00)00346-X Occurrence Handle1:CAS:528:DC%2BD3cXmtVGjsb0%3D Occurrence Handle10962346

    Article  CAS  PubMed  Google Scholar 

  25. PP Van Veldhoven GP Mannaerts (1987) ArticleTitleInorganic and organic phosphate measurements in the nanomolar range. Anal Biochem 161 45–48 Occurrence Handle1:CAS:528:DyaL2sXhtlClsL8%3D Occurrence Handle3578786

    CAS  PubMed  Google Scholar 

  26. D Atlas M Adler (1981) ArticleTitleAlpha-adrenergic antagonists as possible calcium-channel inhibitors. Proc Natl Acad Sci USA Biol Sci 78 1237–1241 Occurrence Handle1:CAS:528:DyaL3MXhsFSisL0%3D

    CAS  Google Scholar 

  27. W Radding SE Jordan RB Hester HC Blair (1999) ArticleTitleIntracellular calcium puffs in osteoclasts. Exp Cell Res 253 689–696 Occurrence Handle10.1006/excr.1999.4714 Occurrence Handle1:CAS:528:DyaK1MXns1amsLY%3D Occurrence Handle10585292

    Article  CAS  PubMed  Google Scholar 

  28. KD Ryder RL Duncan (2000) ArticleTitleParathyroid hormone modulates the response of osteoblast-like cells to mechanical stimulation. Calcif Tissue Int 67 241–246 Occurrence Handle10.1007/s002230001115 Occurrence Handle1:CAS:528:DC%2BD3cXmsVOku7w%3D Occurrence Handle10954779

    Article  CAS  PubMed  Google Scholar 

  29. JJ Lemasters AL Nieminen T Qian LC Trost SP Elmore YXCR Nishimura WE Cascio CA Bradham DA Brenner B Herman (1998) ArticleTitleThe mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy. Biochim Biophys Acta 1366 177–196 Occurrence Handle1:CAS:528:DyaK1cXltFWksb4%3D Occurrence Handle9714796

    CAS  PubMed  Google Scholar 

  30. GW Snedecor WG Cochran (1967) Statistical Methods. The Iowa State University Press Ames IA

    Google Scholar 

  31. MM Kockx J Muhring MW Knaapen GR de Meyer (1998) ArticleTitleRNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis. Am Pathol 152 885–888 Occurrence Handle1:CAS:528:DyaK1cXivVKgtr8%3D

    CAS  Google Scholar 

  32. D Proudfoot JN Skepper L Hegyi MR Bennett CM Shanahan PL Weissberg (2000) ArticleTitleApoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies. Circ Res 87 1055–1062

    Google Scholar 

  33. A Boyde IM Shapiro (1980) ArticleTitleEnergy dispersive X-ray elemental analysis of isolated epiphyseal growth plate chondrocyte fragments. Histochemistry 69 85–94 Occurrence Handle1:CAS:528:DyaL3MXhtlaqtg%3D%3D Occurrence Handle7002886

    CAS  PubMed  Google Scholar 

  34. IA Silver RJ Murrills DJ Etherington (1988) ArticleTitleMicroelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts. Exp Cell Res 175 266–276 Occurrence Handle1:CAS:528:DyaL1cXitF2ru7o%3D Occurrence Handle3360056

    CAS  PubMed  Google Scholar 

  35. NA Thornberry Y Lazebnik (1998) ArticleTitleCaspases: enemies within. Science 281 1312–1316 Occurrence Handle10.1126/science.281.5381.1312 Occurrence Handle1:CAS:528:DyaK1cXlvVegsLs%3D Occurrence Handle9721091

    Article  CAS  PubMed  Google Scholar 

  36. CC Teixeira K Mansfield C Hertkorn H Ischiropoulos IM Shapiro (2001) ArticleTitlePhosphate-induced chondrocyte apoptosis is linked to nitric oxide generation. Am J Physiol Cell Physiol 281 C833–C839 Occurrence Handle1:CAS:528:DC%2BD3MXmvFCgurw%3D Occurrence Handle11502560

    CAS  PubMed  Google Scholar 

  37. H Matsumoto SF Silverton K Debolt IM Shapiro (1991) ArticleTitleSuperoxide dismutase and catalase activities in the growth cartilage, relationship between oxidoreductase activity and chondrocyte maturation. J Bone Miner Res 6 569–574 Occurrence Handle1:CAS:528:DyaK3MXltlaltLc%3D Occurrence Handle1887819

    CAS  PubMed  Google Scholar 

  38. MC Jordani AC Santos IM Prado SA Uyemura C Curti (2000) ArticleTitleFlufenamic acid as an inducer of mitochondrial permeability transition. Mol Cell Biochem 210 153–158 Occurrence Handle10.1023/A:1007185825101 Occurrence Handle1:CAS:528:DC%2BD3cXmtVWgt7w%3D Occurrence Handle10976768

    Article  CAS  PubMed  Google Scholar 

  39. TP Dalton HG Shertzer A Puga (1999) ArticleTitleRegulation of gene expression by reactive oxygen. Ann Rev Pharmacol Toxicol 39 67–101 Occurrence Handle10.1146/annurev.pharmtox.39.1.67 Occurrence Handle1:CAS:528:DyaK1MXjtVejt7w%3D

    Article  CAS  Google Scholar 

  40. CK Sen L Packer (1996) ArticleTitleAntioxidant and redox regulation of gene transcription. FASEB J 10 709–720 Occurrence Handle1:CAS:528:DyaK28Xjtl2gt74%3D Occurrence Handle8635688

    CAS  PubMed  Google Scholar 

  41. W Jr Davis Z Ronai KD Tew (2001) ArticleTitleCellular thiols and reactive oxygen species in drug-induced apoptosis. J Pharmacol Exp Ther 296 1–6 Occurrence Handle1:CAS:528:DC%2BD3MXosVSq Occurrence Handle11123355

    CAS  PubMed  Google Scholar 

  42. LK Moran JM Gutteridge GJ Quinlan (2001) ArticleTitleThiols in cellular redox signalling and control. Curr Med Chem 8 763–772 Occurrence Handle1:CAS:528:DC%2BD3MXjsVChtbk%3D Occurrence Handle11375748

    CAS  PubMed  Google Scholar 

  43. S Tan Y Sagara Y Liu P Maher D Schubert (1998) ArticleTitleThe regulation of reactive oxygen species production during programmed cell death. J Cell Biol 141 1423–1432 Occurrence Handle1:CAS:528:DyaK1cXjvFKktr8%3D Occurrence Handle9628898

    CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by NIH grants DE-13319, DE-10875, and DE05748-01, and by NASA Grant NRA 00-HEDS.

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Authors and Affiliations

  1. Abrahamson Cancer Center, School of Medicine, University of Pennsylvania, Philadelphia, PA 11904, USA

    K. Mansfield

  2. Department of Orthopaedic Surgery, Thomas Jefferson Medical College, Philadelphia, PA 19107, USA

    B. Pucci, C. S. Adams & I. M. Shapiro

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  1. K. Mansfield
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  2. B. Pucci
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  3. C. S. Adams
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  4. I. M. Shapiro
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Correspondence to I. M. Shapiro.

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Mansfield, K., Pucci, B., Adams, C. et al. Induction of Apoptosis in Skeletal Tissues: Phosphate-Mediated Chick Chondrocyte Apoptosis is Calcium Dependent . Calcif Tissue Int 73, 161–172 (2003). https://doi.org/10.1007/s00223-002-1056-z

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