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Detection of Apoptosis of Bone Cells In Vitro

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Osteoporosis

Part of the book series: Methods In Molecular Biology™ ((MIMB,volume 455))

Abstract

Studies during the last decade demonstrated that apoptosis is as important as mitosis for the growth and maintenance of the skeleton and provided information on the significance and molecular regulation of apoptosis of bone cells. It is now known that: (1) all osteoclasts die by apoptosis after completing a bone resorption cycle; (2) the majority of osteoblasts also die, whereas the remainder become lining cells or osteocytes; and (3) osteocytes, although long-living cells, also can die prematurely. Furthermore, mounting evidence indicates that systemic hormones, local growth factors, cytokines, and pharmacological agents, as well as mechanical forces regulate the rate of bone cell apoptosis. This chapter summarizes the methods developed in the last few years to examine apoptosis of cultured bone cells and identify the signaling pathways and molecules involved in apoptosis regulation by diverse skeletal stimuli.

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References

  1. 1. Lecka-Czernik, B., Gubrij, I., Moerman, E. A., et al. (1999) Inhibition of Osf2/Cbfa1 expres sion and terminal osteoblast differentiation by PPAR-gamma 2. J Cell Biochem 74, 357–371.

    Article  CAS  PubMed  Google Scholar 

  2. 2. Bellido, T., Ali, A. A., Plotkin, L. I., et al. (2003) Proteasomal degradation of Runx2 shortens parathyroid hormone-induced anti-apoptotic signaling in osteoblasts. A putative explanation for why intermittent administration is needed for bone anabolism. J Biol Chem 278, 50259–50272.

    Article  CAS  PubMed  Google Scholar 

  3. 3. Kato, Y. , Windle, J. J., Koop, B. A., et al. (1997) Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Min Res 12, 2014–2023.

    Article  CAS  Google Scholar 

  4. 4. Hsu, H., Lacey, D. L., Dunstan, C. R., et al. (1999) Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand. Proc Natl Acad Sci U S A 96, 3540–3545.

    Article  CAS  PubMed  Google Scholar 

  5. 5. Huang, L., Xu, J., Wood, D. J., et al. (2000) Gene expression of osteoprotegerin ligand, osteo-protegerin, and receptor activator of NF-kappaB in giant cell tumor of bone: possible involve ment in tumor cell-induced osteoclast-like cell formation. Am J Pathol 156, 761–767.

    Article  CAS  PubMed  Google Scholar 

  6. 6. Jilka, R. L., Weinstein, R. S., Bellido, T., et al. (1999) Increased bone formation by prevention of osteoblast apoptosis with parathyroid hormone. J Clin Invest 104, 439–446.

    Article  CAS  PubMed  Google Scholar 

  7. 7. Plotkin, L. I., Weinstein, R. S., Parfitt, A. M., et al. (1999) Prevention of osteocyte and oste-oblast apoptosis by bisphosphonates and calcitonin. J Clin Invest 104, 1363–1374.

    Article  CAS  PubMed  Google Scholar 

  8. 8. Selander, K. S., Harkonen, P. L., Valve, E., et al. (1996) Calcitonin promotes osteoclast sur vival in vitro. Mol Cell Endocrinol 122, 119–129.

    Article  CAS  PubMed  Google Scholar 

  9. 9. Chen, J. R., Plotkin, L. I., Aguirre, J. I., et al. (2005) Transient versus sustained phosphoryla-tion and nuclear accumulation of ERKs underlie anti- versus pro-apoptotic effects of estrogens. J Biol Chem 280, 4632–4638.

    Article  CAS  PubMed  Google Scholar 

  10. 10. Kousteni, S., Chen, J. R., Bellido, T., et al. (2002) Reversal of bone loss in mice by nongeno-tropic signaling of sex steroids. Science 298, 843–846.

    Article  CAS  PubMed  Google Scholar 

  11. 11. Kousteni, S., Han, L., Chen, J. R., et al. (2003) Kinase-mediated regulation of common tran scription factors accounts for the bone-protective effects of sex steroids. J Clin Invest 111, 1651–1664.

    CAS  PubMed  Google Scholar 

  12. 12. Kousteni, S., Bellido, T., Plotkin, L. I., et al. (2001) Nongenotropic, sex-nonspecific signaling through the estrogen or androgen receptors: dissociation from transcriptional activity. Cell 104, 719–730.

    CAS  PubMed  Google Scholar 

  13. 13. Plotkin, L. I., Aguirre, J. I., Kousteni, S., et al. (2005) Bisphosphonates and estrogens inhibit osteocyte apoptosis via distinct molecular mechanisms downstream of ERK activation. J Biol Chem 280, 7317–7325.

    Article  CAS  PubMed  Google Scholar 

  14. 14. Vertino, A. M., Bula, C. M., Chen, J. R., et al. (2005) Nongenotropic, anti-apoptotic signaling of 1alpha,25(OH)2-vitamin D3 and analogs through the ligand binding domain of the vitamin D receptor in osteoblasts and osteocytes. Mediation by Src, phosphatidylinositol 3-, and JNK kinases. J Biol Chem 280, 14130–14137.

    Article  CAS  PubMed  Google Scholar 

  15. 15. Bellido, T., O'Brien, C. A., Roberson, P. K., et al. (1998) Transcriptional activation of the p21(WAF1,CIP1,SDI1) gene by interleukin-6 type cytokines. A prerequisite for their pro-differentiating and anti-apoptotic effects on human osteoblastic cells. J Biol Chem 273, 21137–21144.

    Article  CAS  PubMed  Google Scholar 

  16. 16. Bodine, P. V., Billiard, J., Moran, R. A., et al. (2005) The Wnt antagonist secreted frizzled-related protein-1 controls osteoblast and osteocyte apoptosis. J Cell Biochem 96, 1212–1230.

    Article  CAS  PubMed  Google Scholar 

  17. 17. Karsdal, M. A., Larsen, L., Engsig, M. T., et al. (2002) Matrix metalloproteinase-dependent activation of latent transforming growth factor-beta controls the conversion of osteoblasts into osteocytes by blocking osteoblast apoptosis. J Biol Chem 277, 44061–44067.

    Article  CAS  PubMed  Google Scholar 

  18. 18. Fuller, K., Lean, J. M., Bayley, K. E., et al. (2000) A role for TGFbeta(1) in osteoclast differ entiation and survival. J Cell Sci 113, 2445–2453.

    CAS  PubMed  Google Scholar 

  19. 19. Hill, P. A., Tumber, A., Meikle, M. C. (1997) Multiple extracellular signals promote osteoblast survival and apoptosis. Endocrinology 138, 3849–3858.

    Article  CAS  PubMed  Google Scholar 

  20. 20. Plotkin, L. I., Manolagas, S. C., Bellido, T. (2002) Transduction of cell survival signals by connexin-43 hemichannels. J Biol Chem 277, 8648–8657.

    Article  CAS  PubMed  Google Scholar 

  21. 21. Plotkin, L. I., Manolagas, S. C., Bellido, T. (2006) Dissociation of the pro-apoptotic effects of bisphosphonates on osteoclasts from their anti-apoptotic effects on osteoblasts/osteocytes with novel analogs. Bone 39, 443–452.

    Article  CAS  PubMed  Google Scholar 

  22. 22. Weinstein, R. S., Chen, J. R., Powers, C. C., et al. (2002) Promotion of osteoclast survival and antagonism of bisphosphonate-induced osteoclast apoptosis by glucocorticoids. J Clin Invest 109, 1041–1048.

    CAS  PubMed  Google Scholar 

  23. 23. Kogianni, G., Mann, V. , Ebetino, F., et al. (2004) Fas/CD95 is associated with glucocorticoid-induced osteocyte apoptosis. Life Sci 75, 2879–2895.

    Article  CAS  PubMed  Google Scholar 

  24. 24. Ahuja, S. S., Zhao, S., Bellido, T., et al. (2003) CD40 ligand blocks apoptosis induced by tumor necrosis factor alpha, glucocorticoids, and etoposide in osteoblasts and the osteocyte-like cell line murine long bone osteocyte-Y4. Endocrinology 144, 1761–1769.

    Article  CAS  PubMed  Google Scholar 

  25. 25. Almeida, M., Han, L., Bellido, T., et al. (2005) Wnt proteins prevent apoptosis of both uncom mitted osteoblast progenitors and differentiated osteoblasts by beta-catenin-dependent and -independent signaling cascades involving Src/ERK and phosphatidylinositol 3-kinase/AKT. J Biol Chem 280, 41342–41351.

    Article  CAS  PubMed  Google Scholar 

  26. 26. Plotkin, L. I., Mathov, I., Aguirre, J. I., et al. (2005) Mechanical stimulation prevents osteo-cyte apoptosis: requirement of integrins, Src kinases and ERKs. Am J Physiol Cell Physiol 289, C633–C643.

    Article  CAS  PubMed  Google Scholar 

  27. 27. Fatokun, A. A., Stone, T. W., Smith, R. A. (2006) Hydrogen peroxide-induced oxidative stress in MC3T3-E1 cells: the effects of glutamate and protection by purines. Bone 39, 542–551.

    Article  CAS  PubMed  Google Scholar 

  28. 28. Park, B. G., Yoo, C. I., Kim, H. T., et al. (2005) Role of mitogen-activated protein kinases in hydrogen peroxide-induced cell death in osteoblastic cells. Toxicology 215, 115–125.

    Article  CAS  PubMed  Google Scholar 

  29. 29. Bellido, T., Huening, M., Raval-Pandya, M., et al. (2000) Calbindin-D28k is expressed in osteoblastic cells and suppresses their apoptosis by inhibiting caspase-3 activity. J Biol Chem 275, 26328–26332.

    Article  CAS  PubMed  Google Scholar 

  30. 30. Liu, Y. , Porta, A., Peng, X., et al. (2004) Prevention of glucocorticoid-induced apoptosis in osteocytes and osteoblasts by calbindin-D28k. J Bone Miner Res 19, 479–490.

    Article  CAS  PubMed  Google Scholar 

  31. 31. Pavalko, F. M., Gerard, R. L., Ponik, S. M., et al. (2003) Fluid shear stress inhibits TNF-alpha-induced apoptosis in osteoblasts: a role for fluid shear stress-induced activation of PI3-kinase and inhibition of caspase-3. J Cell Physiol 194, 194–205.

    Article  CAS  PubMed  Google Scholar 

  32. 32. Bakker, A., Klein-Nulend, J., Burger, E. (2004) Shear stress inhibits while disuse promotes osteocyte apoptosis. Biochem Biophys Res Commun 320, 1163–1168.

    Article  CAS  PubMed  Google Scholar 

  33. 33. Kitase, Y. , Jiang, J. X., Johnson, M. L., et al. (2006) The anti-apoptotic effects of mechanical strain on osteocytes are mediated by PGE2 and monocyte chemotactic protein-3 (MCP-3): selective protection by MCP-3 against glucocorticoid (GC), but not TNF-a induced apoptosis. J Bone Miner Res 21, S48.

    Google Scholar 

  34. 34. Alessi, D. R., Cuenda, A., Cohen, P., et al. (1995) PD 098059 is a specific inhibitor of the activation of mitogen-activated protein kinase kinase in vitro and in vivo. J Biol Chem 270, 27489–27494.

    Article  CAS  PubMed  Google Scholar 

  35. 35. Nicholson, D. W., Ali, A., Thornberry, N. A., et al. (1995) Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature 376, 37–43.

    Article  CAS  PubMed  Google Scholar 

  36. 36. Jilka, R. L., Weinstein, R. S., Bellido, T., et al. (1998) Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Min Res 13, 793–802.

    Article  CAS  Google Scholar 

  37. 37. Frisch, S. M., Vuori, K., Ruoslahti, E., et al. (1996) Control of adhesion-dependent cell sur vival by focal adhesion kinase. J Cell Biol 134, 793–799.

    Article  CAS  PubMed  Google Scholar 

  38. 38. Murakami, T., Yamamoto, M., Ono, K., et al. (1998) Transforming growth factor-beta1 increases mRNA levels of osteoclastogenesis inhibitory factor in osteoblastic/stromal cells and inhibits the survival of murine osteoclast-like cells. Biochem Biophys Res Commun 252, 747–752.

    Article  CAS  PubMed  Google Scholar 

  39. 39. Kameda, T., Mano, H., Yuasa, T., et al. (1997) Estrogen inhibits bone resorption by directly inducing apoptosis of the bone-resorbing osteoclasts. J Exp Med 186, 489–495.

    Article  CAS  PubMed  Google Scholar 

  40. 40. Chua, C. C., Chua, B. H., Chen, Z., et al. (2003) Dexamethasone induces caspase activation in murine osteoblastic MC3T3-E1 cells. Biochim Biophys Acta 1642, 79–85.

    Article  CAS  PubMed  Google Scholar 

  41. 41. Mahajan, N. P., Harrison-Shostak, D. C., Michaux, J., et al. (1999) Novel mutant green fluo rescent protein protease substrates reveal the activation of specific caspases during apoptosis. Chem Biol 6, 401–409.

    Article  CAS  PubMed  Google Scholar 

  42. 42. Tyas, L., Brophy, V. A., Pope, A., et al. (2000) Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer. EMBO Rep 1, 266–270.

    Article  CAS  PubMed  Google Scholar 

  43. 43. Mohr, S., McCormick, T. S., Lapetina, E. G. (1998) Macrophages resistant to endogenously generated nitric oxide-mediated apoptosis are hypersensitive to exogenously added nitric oxide donors: dichotomous apoptotic response independent of caspase 3 and reversal by the mitogen-activated protein kinase kinase (MEK) inhibitor PD 098059. Proc Natl Acad Sci U S A 95, 5045–5050.

    Article  CAS  PubMed  Google Scholar 

  44. 44. O'Brien, C. A., Jia, D., Plotkin, L. I., et al. (2004) Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology 145, 1835–1841.

    Article  PubMed  Google Scholar 

  45. 45. Hiroi-Furuya, E., Kameda, T., Hiura, K., et al. (1999) Etidronate (EHDP) inhibits osteoclastic-bone resorption, promotes apoptosis and disrupts actin rings in isolate-mature osteoclasts. Calcif Tissue Int 64, 219–223.

    Article  CAS  PubMed  Google Scholar 

  46. 46. Hughes, D. E., Wright, K. R., Uy, H. L., et al. (1995) Bisphosphonates promote apoptosis in murine osteoclasts in vitro and in vivo. J Bone Min Res 10, 1478–1487.

    Article  CAS  Google Scholar 

  47. 47. Hughes, D. E., Dai, A., Tiffee, J. C., et al. (1996) Estrogen promotes apoptosis of murine osteoclasts mediated by TGF-beta. Nat Med 2, 1132–1136.

    Article  CAS  PubMed  Google Scholar 

  48. 48. Chang, C. C., Liao, Y. S., Lin, Y. L., et al. (2006) Nitric oxide protects osteoblasts from oxida-tive stress-induced apoptotic insults via a mitochondria-dependent mechanism. J Orthop Res 24, 1917–1925.

    Article  CAS  PubMed  Google Scholar 

  49. 49. Wiren, K. M., Toombs, A. R., Semirale, A. A., et al. (2006) Osteoblast and osteocyte apoptosis associated with androgen action in bone: requirement of increased Bax/Bcl-2 ratio. Bone 38, 637–651.

    Article  CAS  PubMed  Google Scholar 

  50. 50. Ho, W. P., Chen, T. L., Chiu, W. T., et al. (2005) Nitric oxide induces osteoblast apoptosis through a mitochondria-dependent pathway. Ann NY Acad Sci 1042, 460–470.

    Article  CAS  PubMed  Google Scholar 

  51. 51. Kim, G. S., Hong, J. S., Kim, S. W., et al. (2003) Leptin induces apoptosis via ERK/cPLA2/ cytochrome c pathway in human bone marrow stromal cells. J Biol Chem 278, 21920–21929.

    Article  CAS  PubMed  Google Scholar 

  52. 52. Oursler, M. J., Bradley, E. W., Elfering, S. L., et al. (2005) Native, not nitrated, cytochrome c and mitochondrial-derived hydrogen peroxide drive osteoclast apoptosis. Am J Physiol Cell Physiol 288, C156–C168.

    CAS  PubMed  Google Scholar 

  53. 53. Wu, X., McKenna, M. A., Feng, X., et al. (2003) Osteoclast apoptosis: the role of Fas in vivo and in vitro. Endocrinology 144, 5545–5555.

    Article  CAS  PubMed  Google Scholar 

  54. 54. Qin, Y. J., Zhang, Z. L., Yu, L. Y. , et al. (2006) A20 overexpression under control of mouse osteocalcin promoter in MC3T3-E1 cells inhibited tumor necrosis factor-alpha-induced apop-tosis. Acta Pharmacol Sin 27, 1231–1237.

    Article  CAS  PubMed  Google Scholar 

  55. 55. Contardi, E., Palmisano, G. L., Tazzari, P. L., et al. (2005) CTLA-4 is constitutively expressed on tumor cells and can trigger apoptosis upon ligand interaction. Int J Cancer 117, 538–550.

    Article  CAS  PubMed  Google Scholar 

  56. 56. Postiglione, L., Di, D. G., Ramaglia, L., et al. (2003) Behavior of SaOS-2 cells cultured on different titanium surfaces. J Dent Res 82, 692–696.

    Article  CAS  PubMed  Google Scholar 

  57. 57. Alikhani, M., Alikhani, Z., Boyd, C., et al. (2007) Advanced glycation end products stimulate osteoblast apoptosis via the MAP kinase and cytosolic apoptotic pathways. Bone 40, 345–353.

    Article  CAS  PubMed  Google Scholar 

  58. 58. Kim, H. J., Zhao, H., Kitaura, H., et al. (2006) Glucocorticoids suppress bone formation via the osteoclast. J Clin Invest 116, 2152–2160.

    Article  CAS  PubMed  Google Scholar 

  59. 59. Mogi, M., Togari, A. (2003) Activation of caspases is required for osteoblastic differentiation. J Biol Chem 278, 47477–47482.

    Article  CAS  PubMed  Google Scholar 

  60. 60. Wang, Y.H., Liu, Y., Rowe, D.W. (2007) Effects of transient PTH on early proliferation, apop-tosis, and subsequent differentiation of osteoblast in calvarial osteoblast cultures. Am J Physiol Endocrinol Metab 292, E594–E603.

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Division of Endocrinology & Metabolism and Center for Osteoporosis and Metabolic Bone Diseases, University of Arkansas for Medical Sciences, Little Rock, AR

    Teresita Bellido & Lilian I. Plotkin

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  1. Teresita Bellido
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  2. Lilian I. Plotkin
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Editors and Affiliations

  1. Department of Orthopedic Surgery, Mayo Clinic, Rochester, MN, USA

    Jennifer J. Westendorf

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© 2008 Humana Press, a part of Springer Science+Business Media, LLC

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Bellido, T., Plotkin, L.I. (2008). Detection of Apoptosis of Bone Cells In Vitro . In: Westendorf, J.J. (eds) Osteoporosis. Methods In Molecular Biology™, vol 455. Humana Press. https://doi.org/10.1007/978-1-59745-104-8_4

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  • DOI: https://doi.org/10.1007/978-1-59745-104-8_4

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-58829-828-7

  • Online ISBN: 978-1-59745-104-8

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