Calcified Tissue International

, Volume 73, Issue 2, pp 161–172 | Cite as

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

  • K. Mansfield
  • B. Pucci
  • C. S. Adams
  • I. M. ShapiroEmail author
Laboratory Investigations


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.


Chondrocyte Apoptosis Phosphate Calcium ROS 



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


  1. 1.
    Bronckers, ALJJ, Goei, W, Luo, G, Karsenty, G, D’Souza, RN, Lyaruu, DM, Burger, EH 1996DNA fragmentation during bone formation in neonatal rodents assessed by transferase-mediated end labeling.J Bone Miner Res1112811291PubMedGoogle Scholar
  2. 2.
    Farnum, CE, Wilsman, NJ 1987Morphologic stages of the terminal hypertrophic chondrocyte of growth plate cartilage.Anat Rec219221232PubMedGoogle Scholar
  3. 3.
    Farnum, CE, Wilsman, NJ 1989Cellular turnover at the chondro–osseous junction of growth plate cartilage: Analysis by serial sections at the light microscopical level.J Orthop Res7654666PubMedGoogle Scholar
  4. 4.
    Farnum, CE, Wilsman, NJ 1989Condensation of hypertrophic chondrocytes at the condro–osseous junction of growth plate cartilage in yucatan swine: Relationship to long bone growth.Am J Anat186346358PubMedGoogle Scholar
  5. 5.
    Gibson, GJ, Kohler, WJ, Schaffler, MB 1995Chondrocyte apoptosis in endochondrial ossification of chick sterna.Dev Dyn203468476PubMedGoogle Scholar
  6. 6.
    Hatori, M, Klatte, KJ, Teixeira, CC, Shapiro, IM 1995End labeling studies of fragmented DNA in the avian growth plate: evidence of apoptosisin terminally differentiated chondrocytes.J Bone Miner Res1019601968PubMedGoogle Scholar
  7. 7.
    Ishizaki, Y, Burne, JF, Raff, MC 1994Autocrine signals enable chondrocytes to survive in culture.J Cell Biol12610691077PubMedGoogle Scholar
  8. 8.
    Ishizaki, Y, Cheng, L, Mudge, AW, Raff, MC 1995Programmed cell death by default in embryonic cells, fibroblasts, and cancer cells.Mol Biol Cell614431458PubMedGoogle Scholar
  9. 9.
    Mitrovic, D, Quintero, M, Stankovic, A, Ryckewaert, A 1983Cell density of adult human femoral condylar articular cartilage. Joints with normal and fibrillated surfaces.Lab Invest49309316PubMedGoogle Scholar
  10. 10.
    Quintero, M, Mitrovic, D, Stankovic, A, de Seze, S, Miravet, L, Ryckewaert, A 1984Aspects Cellulaires du viellissement du cartilage articulaire I. Cartilage condylien a surface normale, preleve dans les genoux normaux.Rev Rhum51375379PubMedGoogle Scholar
  11. 11.
    Quintero, M, Mitrovic, D, Stankovic, A, de Seze, S, Miravet, L, Ryckewaert, A 1984Aspects cellulaires du viellissement du cartilage articulaire II. Cartilage condylien a surface fissuree preleve dans les genoux “normaux” et “arthrosiques.”Rev Rhum51445449PubMedGoogle Scholar
  12. 12.
    Vignon, E, Arlot, M, Patricot, LM, Vignon, G 1976The cell density of human femoral head cartilage.Clin Orthop121303308PubMedGoogle Scholar
  13. 13.
    Hochberg, MC, Lethbridge–Cejku, M, Scott Jr, WW, Plato, CC, Tobin, JD 1993Obesity and osteoarthritis of the hands in women.Osteoarthritis Cartilage1129135PubMedGoogle Scholar
  14. 14.
    Lethbridge–Cejku, M, Tobin, JD, Scott Jr, WW, Reichle, R, Plato, CC, Hochberg, MC 1994The 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)6353357Google Scholar
  15. 15.
    Adams, CS, Horton, WE 1998Chondrocyte apoptosis increases with age in the articular cartilage of adult animals.Anat Rec250418425CrossRefPubMedGoogle Scholar
  16. 16.
    Mansfield, K, Rajpurohit, R, Shapiro, IM 1999Extracellular phosphate ions cause apoptosis of terminally differentiated epiphyseal chondrocytes.J Cell Physiol179276286CrossRefPubMedGoogle Scholar
  17. 17.
    Mansfield, K, Teixeira, CC, Adams, CS, Shapiro, IM 2001Phosphate ions mediate chondrocyte apoptosis through a plasma membrane transporter mechanism.Bone2818CrossRefPubMedGoogle Scholar
  18. 18.
    Rajpurohit, R, Mansfield, K, Ohyama, K, Ewert, D, Shapiro, IM 1999Chondrocyte death is linked to development of a mitochondrial membrane permeability transition in the growth plate.J Cell Physiol179287296CrossRefPubMedGoogle Scholar
  19. 19.
    Adams, CS, Mansfield, K, Perlot, RL, Shapiro, IM 2001Matrix regulation of skeletal cell apoptosis—Role of calcium and phosphate ions.J Biol Chem2762031620322CrossRefPubMedGoogle Scholar
  20. 20.
    Rajpurohit, R, Koch, CJ, Tao, Z, Teixeira, CM, Shapiro, IM 1996Adaptation of chondrocytes to low oxygen tension: relationship between hypoxia and cellular metabolism.J Cell Physiol168424432CrossRefPubMedGoogle Scholar
  21. 21.
    Oshima, O, Leboy, PS, McDonald, SA, Tuan, RS, Shapiro, IM 1989Developmental expression of genes in chick growth cartilage detected by in situ hybridization.Calcif Tissue Int45182192PubMedGoogle Scholar
  22. 22.
    McGahon, AJ, Martin, SJ, Bissonnette, RP, Mahboubi, A, Shi, Y, Mogil, RJ, Nishioka, WK, Green, DR 1995The end of the (cell) line: methods for the study of apoptosis in vitro.Meth Cell Biol46153185Google Scholar
  23. 23.
    Baxter, GD, Collins, RJ, Harmon, BV, Kumar, S, Prentice, RL, Smith, PJ, Lavin, MF 1989Cell death by apoptosis in acute leukaemia.J Pathol158123129PubMedGoogle Scholar
  24. 24.
    Meleti, Z, Shapiro, IM, Adams, CS 2000Inorganic phosphate induces apoptosis of osteoblast-like cells in culture.Bone27359366CrossRefPubMedGoogle Scholar
  25. 25.
    Van Veldhoven, PP, Mannaerts, GP 1987Inorganic and organic phosphate measurements in the nanomolar range.Anal Biochem1614548PubMedGoogle Scholar
  26. 26.
    Atlas, D, Adler, M 1981Alpha-adrenergic antagonists as possible calcium-channel inhibitors.Proc Natl Acad Sci USA Biol Sci7812371241Google Scholar
  27. 27.
    Radding, W, Jordan, SE, Hester, RB, Blair, HC 1999Intracellular calcium puffs in osteoclasts.Exp Cell Res253689696CrossRefPubMedGoogle Scholar
  28. 28.
    Ryder, KD, Duncan, RL 2000Parathyroid hormone modulates the response of osteoblast-like cells to mechanical stimulation.Calcif Tissue Int67241246CrossRefPubMedGoogle Scholar
  29. 29.
    Lemasters, JJ, Nieminen, AL, Qian, T, Trost, LC, Elmore, SP, Nishimura, YXCR, Cascio, WE, Bradham, CA, Brenner, DA, Herman, B 1998The mitochondrial permeability transition in cell death: a common mechanism in necrosis, apoptosis and autophagy.Biochim Biophys Acta1366177196PubMedGoogle Scholar
  30. 30.
    Snedecor, GW, Cochran, WG 1967Statistical Methods.The Iowa State University PressAmes IAGoogle Scholar
  31. 31.
    Kockx, MM, Muhring, J, Knaapen, MW, de Meyer, GR 1998RNA synthesis and splicing interferes with DNA in situ end labeling techniques used to detect apoptosis.Am Pathol152885888Google Scholar
  32. 32.
    Proudfoot, D, Skepper, JN, Hegyi, L, Bennett, MR, Shanahan, CM, Weissberg, PL 2000Apoptosis regulates human vascular calcification in vitro: evidence for initiation of vascular calcification by apoptotic bodies.Circ Res8710551062Google Scholar
  33. 33.
    Boyde, A, Shapiro, IM 1980Energy dispersive X-ray elemental analysis of isolated epiphyseal growth plate chondrocyte fragments.Histochemistry698594PubMedGoogle Scholar
  34. 34.
    Silver, IA, Murrills, RJ, Etherington, DJ 1988Microelectrode studies on the acid microenvironment beneath adherent macrophages and osteoclasts.Exp Cell Res175266276PubMedGoogle Scholar
  35. 35.
    Thornberry, NA, Lazebnik, Y 1998Caspases: enemies within.Science28113121316CrossRefPubMedGoogle Scholar
  36. 36.
    Teixeira, CC, Mansfield, K, Hertkorn, C, Ischiropoulos, H, Shapiro, IM 2001Phosphate-induced chondrocyte apoptosis is linked to nitric oxide generation.Am J Physiol Cell Physiol281C833C839PubMedGoogle Scholar
  37. 37.
    Matsumoto, H, Silverton, SF, Debolt, K, Shapiro, IM 1991Superoxide dismutase and catalase activities in the growth cartilage, relationship between oxidoreductase activity and chondrocyte maturation.J Bone Miner Res6569574PubMedGoogle Scholar
  38. 38.
    Jordani, MC, Santos, AC, Prado, IM, Uyemura, SA, Curti, C 2000Flufenamic acid as an inducer of mitochondrial permeability transition.Mol Cell Biochem210153158CrossRefPubMedGoogle Scholar
  39. 39.
    Dalton, TP, Shertzer, HG, Puga, A 1999Regulation of gene expression by reactive oxygen.Ann Rev Pharmacol Toxicol3967101CrossRefGoogle Scholar
  40. 40.
    Sen, CK, Packer, L 1996Antioxidant and redox regulation of gene transcription.FASEB J10709720PubMedGoogle Scholar
  41. 41.
    Davis, W Jr, Ronai, Z, Tew, KD 2001Cellular thiols and reactive oxygen species in drug-induced apoptosis.J Pharmacol Exp Ther29616PubMedGoogle Scholar
  42. 42.
    Moran, LK, Gutteridge, JM, Quinlan, GJ 2001Thiols in cellular redox signalling and control.Curr Med Chem8763772PubMedGoogle Scholar
  43. 43.
    Tan, S, Sagara, Y, Liu, Y, Maher, P, Schubert, D 1998The regulation of reactive oxygen species production during programmed cell death.J Cell Biol14114231432PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • K. Mansfield
    • 1
  • B. Pucci
    • 2
  • C. S. Adams
    • 2
  • I. M. Shapiro
    • 2
    Email author
  1. 1.Abrahamson Cancer Center, School of MedicineUniversity of Pennsylvania, Philadelphia, PA 11904USA
  2. 2.Department of Orthopaedic SurgeryThomas Jefferson Medical College, Philadelphia, PA 19107USA

Personalised recommendations