Skip to main content

Advertisement

SpringerLink
Log in

Bone-Targeted Overexpression of Bcl-2 Increases Osteoblast Adhesion and Differentiation and Inhibits Mineralization In Vitro

  • Published:
Calcified Tissue International Aims and scope Submit manuscript

Abstract

Apoptosis is a process important for the development and homeostasis of self-renewing tissues, including bone. However, little is known about the function of Bcl-2, a key player of apoptosis, in the regulation of osteoblast activity. Ex vivo cultures of osteoblasts from Col2.3Bcl-2 mice, in which human Bcl-2 was targeted to bone by the 2.3 kb fragment of the type I collagen promoter, were used to study the effect of Bcl-2 in osteoblasts. During 35 days of culture, hBcl-2 expression increased without any effect on endogenous mouse Bcl-2 and Bax expression. Adhesion of transgenic (TG) osteoblasts was twofold more than that of wild-type (WT) cells, with significantly higher expression of integrins α1, α2, and α5 but similar levels of αv and β1 relative to WT cells. Proliferation of osteoblasts was not affected. Overexpression of hBcl-2 promoted the differentiation of osteoblasts, as shown by increased message levels of alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin in the TG compared to WT cells throughout the culture period. The two transcription factors essential for osteoblast differentiation, core binding factor alpha 1 (Cbfa-1) and osterix, had significantly higher expression in TG than WT cells during the early culture period. ß-Catenin, a central player in the canonical Wnt pathway, also had higher expression in TG than WT cultures. Mineralization was significantly decreased in TG cultures, with less osteoblast apoptosis, compared to WT. Thus, Bcl-2 seems to have multiple roles in modulating osteoblast activities.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1.
Fig. 2.
Fig. 3.
Fig. 4.
Fig. 5.
Fig. 6.
Fig. 7.

Similar content being viewed by others

References

  1. Tsujimoto Y, Finger LR, Yunis J, Nowell PC, Croce CM (1984) Cloning of the chromosome breakpoint of neoplastic B cells with the t(14;18) chromosome translocation. Science 226:1097–1099

    Article  PubMed  CAS  Google Scholar 

  2. Kroemer G (1997) The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 3:614–620

    Article  PubMed  CAS  Google Scholar 

  3. Green DR, Reed JC (1998) Mitochondria and apoptosis. Science 281:1309–1312

    Article  PubMed  CAS  Google Scholar 

  4. Adams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281:1322–1326

    Article  PubMed  CAS  Google Scholar 

  5. Cory S, Strasser A, Jacks T, Corcoran LM, Metz T, Harris AW, Adams JM (1994) Enhanced cell survival and tumorigenesis. Cold Spring Harb Symp Quant Biol 59:365–375

    PubMed  CAS  Google Scholar 

  6. Cory S (1995) Regulation of lymphocyte survival by the bcl-2 gene family. Annu Rev Immunol 13:513–543

    Article  PubMed  CAS  Google Scholar 

  7. Strasser A, Huang DC, Vaux DL (1997) The role of the bcl-2/ced-9 gene family in cancer and general implications of defects in cell death control for tumorigenesis and resistance to chemotherapy. Biochim Biophys Acta 1333:F151–F178

    PubMed  CAS  Google Scholar 

  8. Yang E, Korsmeyer SJ (1996) Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 88:386–401

    PubMed  CAS  Google Scholar 

  9. Chao DT, Korsmeyer SJ (1998) BCL-2 family: regulators of cell death. Annu Rev Immunol 16:395–419

    Article  PubMed  CAS  Google Scholar 

  10. Mocetti P, Silvestrini G, Ballanti P, Patacchioli FR, Di Grezia R, Angelucci L, Bonucci E (2001) Bcl-2 and Bax expression in cartilage and bone cells after high-dose corticosterone treatment in rats. Tissue Cell 33:1–7

    Article  PubMed  CAS  Google Scholar 

  11. Boot-Handford RP, Michaelidis TM, Hillarby MC, Zambelli A, Denton J, Hoyland JA, Freemont AJ, Grant ME, Wallis GA (1998) The bcl-2 knockout mouse exhibits marked changes in osteoblast phenotype and collagen deposition in bone as well as a mild growth plate phenotype. Int J Exp Pathol 79:329–335

    Article  PubMed  CAS  Google Scholar 

  12. Amling M, Neff L, Tanaka S, Inoue D, Kuida K, Weir E, Philbrick WM, Broadus AE, Baron R (1997) Bcl-2 lies downstream of parathyroid hormone-related peptide in a signaling pathway that regulates chondrocyte maturation during skeletal development. J Cell Biol 136:205–213

    Article  PubMed  CAS  Google Scholar 

  13. Jilka RL, Weinstein RS, Bellido T, Parfitt AM, Manolagas SC (1998) Osteoblast programmed cell death (apoptosis): modulation by growth factors and cytokines. J Bone Miner Res 13:793–802

    Article  PubMed  CAS  Google Scholar 

  14. Gronowicz GA, McCarthy MB (1995) Glucocorticoids inhibit the attachment of osteoblasts to bone extracellular matrix proteins and decrease beta1-integrin levels. Endocrinology 136:598–608

    Article  PubMed  CAS  Google Scholar 

  15. Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11–25

    Article  PubMed  CAS  Google Scholar 

  16. Ruoslahti E (1991) Integrins. J Clin Invest 87:1–5

    Article  PubMed  CAS  Google Scholar 

  17. Cowles EA, Brailey LL, Gronowicz GA (2000) Integrin-mediated signaling regulates AP-1 transcription factors and proliferation in osteoblasts. J Biomed Mater Res 52:725–737

    Article  PubMed  CAS  Google Scholar 

  18. Gronowicz GA, Derome ME (1994) Synthetic peptide containing Arg-Gly-Asp inhibits bone formation and resorption in a mineralizing organ culture system of fetal rat parietal bones. J Bone Miner Res 9:193–201

    PubMed  CAS  Google Scholar 

  19. Lee BH, Ruoslahti E (2005) α5β1 integrin stimulates Bcl-2 expression and cell survival through Akt, focal adhesion kinase, and Ca2+/calmodulin-dependent protein kinase IV. J Cell Biochem 95:1214–1223

    Article  PubMed  CAS  Google Scholar 

  20. Matter ML, Ruoslahti E (2001) A signaling pathway from the α5β1 and αvβ3 integrins that elevates bcl-2 transcription. J Biol Chem 276:27757–27763

    Article  PubMed  CAS  Google Scholar 

  21. Tran NL, Adams DG, Vaillancourt RR, Heimark RL (2002) Signal transduction from N-cadherin increases Bcl-2. Regulation of the phosphatidylinositol 3-kinase/Akt pathway by homophilic adhesion and actin cytoskeletal organization. J Biol Chem 277:32905–32914

    Article  PubMed  CAS  Google Scholar 

  22. Lu PJ, Lu QL, Rughetti A, Taylor-Papadimitriou J (1995) bcl-2 overexpression inhibits cell death and promotes the morphogenesis, but not tumorigenesis of human mammary epithelial cells. J Cell Biol 129:1363–1378

    Article  PubMed  CAS  Google Scholar 

  23. Li L, Backer J, Wong AS, Schwanke EL, Stewart BG, Pasdar M (2003) Bcl-2 expression decreases cadherin-mediated cell-cell adhesion. J Cell Sci 116:3687–3700

    Article  PubMed  CAS  Google Scholar 

  24. Vermeulen K, Berneman ZN, Van Bockstaele DR (2003) Cell cycle and apoptosis. Cell Prolif 36:165–175

    Article  PubMed  CAS  Google Scholar 

  25. Belanger S, Cote M, Lane D, L’Esperance S, Rancourt C, Piche A (2005) Bcl-2 decreases cell proliferation and promotes accumulation of cells in S phase without affecting the rate of apoptosis in human ovarian carcinoma cells. Gynecol Oncol 97:796–806

    Article  PubMed  CAS  Google Scholar 

  26. Linette GP, Li Y, Roth K, Korsmeyer SJ (1996) Cross talk between cell death and cell cycle progression: BCL-2 regulates NFAT-mediated activation. Proc Natl Acad Sci USA 93:9545–9552

    Article  PubMed  CAS  Google Scholar 

  27. Borner C (1996) Diminished cell proliferation associated with the death-protective activity of Bcl-2. J Biol Chem 271:12695–12698

    PubMed  CAS  Google Scholar 

  28. Mazel S, Burtrum D, Petrie HT (1996) Regulation of cell division cycle progression by bcl-2 expression: a potential mechanism for inhibition of programmed cell death. J Exp Med 183:2219–2226

    Article  PubMed  CAS  Google Scholar 

  29. Limana F, Urbanek K, Chimenti S, Quaini F, Leri A, Kajstura J, Nadal-Ginard B, Izumo S, Anversa P (2002) bcl-2 overexpression promotes myocyte proliferation. Proc Natl Acad Sci USA 99:6257–6262

    Article  PubMed  CAS  Google Scholar 

  30. Lu QL, Abel P, Foster CS, Lalani EN (1996) bcl-2: role in epithelial differentiation and oncogenesis. Hum Pathol 27:102–110

    Article  PubMed  CAS  Google Scholar 

  31. Linette GP, Korsmeyer SJ (1994) Differentiation and cell death: lessons from the immune system. Curr Opin Cell Biol 6:809–815

    Article  PubMed  CAS  Google Scholar 

  32. Liang Y, Mirnics ZK, Yan C, Nylander KD, Schor NF (2003) Bcl-2 mediates induction of neural differentiation. Oncogene 22:5515–5518

    Article  PubMed  CAS  Google Scholar 

  33. Weber GF, Menko AS (2005) The canonical intrinsic mitochondrial death pathway has a non-apoptotic role in signaling lens cell differentiation. J Biol Chem 280:22135–22145

    Article  PubMed  CAS  Google Scholar 

  34. Dahm R (1999) Lens fibre cell differentiation - a link with apoptosis? Ophthalmic Res 31:163–183

    Article  PubMed  CAS  Google Scholar 

  35. Harada H, Mitsuyasu T, Seta Y, Maruoka Y, Toyoshima K, Yasumoto S (1998) Overexpression of bcl-2 protein inhibits terminal differentiation of oral keratinocytes in vitro. J Oral Pathol Med 27:11–17

    Article  PubMed  CAS  Google Scholar 

  36. Sanders EJ, Parker E (2003) Retroviral overexpression of bcl-2 in the embryonic chick lens influences denucleation in differentiating lens fiber cells. Differentiation 71:425–433

    Article  PubMed  CAS  Google Scholar 

  37. Sordet O, Rebe C, Plenchette S, Zermati Y, Hermine O, Vainchenker W, Garrido C, Solary E, Dubrez-Daloz L (2002) Specific involvement of caspases in the differentiation of monocytes into macrophages. Blood 100:4446–4453

    Article  PubMed  CAS  Google Scholar 

  38. Komori T (2006) Regulation of osteoblast differentiation by transcription factors. J Cell Biochem

  39. Komori T (2005) Regulation of skeletal development by the Runx family of transcription factors. J Cell Biochem 95:445–453

    Article  PubMed  CAS  Google Scholar 

  40. Nakashima K, Zhou X, Kunkel G, Zhang Z, Deng JM, Behringer RR, de Crombrugghe B (2002) The novel zinc finger-containing transcription factor osterix is required for osteoblast differentiation and bone formation. Cell 108:17–29

    Article  PubMed  CAS  Google Scholar 

  41. Hartmann C (2006) A Wnt canon orchestrating osteoblastogenesis. Trends Cell Biol 16:151–158

    Article  PubMed  CAS  Google Scholar 

  42. Komori T, Yagi H, Nomura S, Yamaguchi A, Sasaki K, Deguchi K, Shimizu Y, Bronson RT, Gao YH, Inada M, Sato M, Okamoto R, Kitamura Y, Yoshiki S, Kishimoto T (1997) Targeted disruption of Cbfa1 results in a complete lack of bone formation owing to maturational arrest of osteoblasts. Cell 89:755–764

    Article  PubMed  CAS  Google Scholar 

  43. Otto F, Thornell AP, Crompton T, Denzel A, Gilmour KC, Rosewell IR, Stamp GW, Beddington RS, Mundlos S, Olsen BR, Selby PB, Owen MJ (1997) Cbfa1, a candidate gene for cleidocranial dysplasia syndrome, is essential for osteoblast differentiation and bone development. Cell 89:765–771

    Article  PubMed  CAS  Google Scholar 

  44. Brault V, Moore R, Kutsch S, Ishibashi M, Rowitch DH, McMahon AP, Sommer L, Boussadia O, Kemler R (2001) Inactivation of the beta-catenin gene by Wnt1-Cre-mediated deletion results in dramatic brain malformation and failure of craniofacial development. Development 128:1253–1264

    PubMed  CAS  Google Scholar 

  45. Day TF, Guo X, Garrett-Beal L, Yang Y (2005) Wnt/beta-catenin signaling in mesenchymal progenitors controls osteoblast and chondrocyte differentiation during vertebrate skeletogenesis. Dev Cell 8:739–750

    Article  PubMed  CAS  Google Scholar 

  46. Hill TP, Spater D, Taketo MM, Birchmeier W, Hartmann C (2005) Canonical Wnt/beta-catenin signaling prevents osteoblasts from differentiating into chondrocytes. Dev Cell 8:727–738

    Article  PubMed  CAS  Google Scholar 

  47. Lynch MP, Capparelli C, Stein JL, Stein GS, Lian JB (1998) Apoptosis during bone-like tissue development in vitro. J Cell Biochem 68:31–49

    Article  PubMed  CAS  Google Scholar 

  48. McCabe LR, Kockx M, Lian J, Stein J, Stein G (1995) Selective expression of fos- and jun-related genes during osteoblast proliferation and differentiation. Exp Cell Res 218:255–262

    Article  PubMed  CAS  Google Scholar 

  49. Gronowicz G, Woodiel FN, McCarthy MB, Raisz LG (1989) In vitro mineralization of fetal rat parietal bones in defined serum-free medium: effect of beta-glycerol phosphate. J Bone Miner Res 4:313–324

    PubMed  CAS  Google Scholar 

  50. Pantschenko AG, Zhang W, Nahounou M, McCarthy MB, Stover ML, Lichtler AC, Clark SH, Gronowicz GA (2005) Effect of osteoblast-targeted expression of bcl-2 in bone: differential response in male and female mice. J Bone Miner Res 20:1414–1429

    Article  PubMed  CAS  Google Scholar 

  51. Cleary ML, Smith SD, Sklar J (1986) Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation. Cell 47:19–28

    Article  PubMed  CAS  Google Scholar 

  52. Wong GL, Cohn DV (1975) Target cells in bone for parathormone and calcitonin are different: enrichment for each cell type by sequential digestion of mouse calvaria and selective adhesion to polymeric surfaces. Proc Natl Acad Sci USA 72:3167–3171

    Article  PubMed  CAS  Google Scholar 

  53. McCarthy TL, Centrella M, Canalis E (1988) Further biochemical and molecular characterization of primary rat parietal bone cell cultures. J Bone Miner Res 3:401–408

    PubMed  CAS  Google Scholar 

  54. Thiede MA, Yoon K, Golub EE, Noda M, Rodan GA (1988) Structure and expression of rat osteosarcoma (ROS 17/2.8) alkaline phosphatase: product of a single copy gene. Proc Natl Acad Sci USA 85:319–323

    Article  PubMed  CAS  Google Scholar 

  55. Genovese C, Rowe D, Kream B (1984) Construction of DNA sequences complementary to rat alpha 1 and alpha 2 collagen mRNA and their use in studying the regulation of type I collagen synthesis by 1,25-dihydroxyvitamin D. Biochemistry 23:6210–6216

    Article  PubMed  CAS  Google Scholar 

  56. Young MF, Ibaraki K, Kerr JM, Lyu MS, Kozak CA (1994) Murine bone sialoprotein (BSP): cDNA cloning, mRNA expression, and genetic mapping. Mamm Genome 5:108–111

    Article  PubMed  CAS  Google Scholar 

  57. Celeste AJ, Rosen V, Buecker JL, Kriz R, Wang EA, Wozney JM (1986) Isolation of the human gene for bone gla protein utilizing mouse and rat cDNA clones. EMBO J 5:1885–1890

    PubMed  CAS  Google Scholar 

  58. Ducy P, Zhang R, Geoffroy V, Ridall AL, Karsenty G (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89:747–754

    Article  PubMed  CAS  Google Scholar 

  59. Kalajzic I, Kalajzic Z, Kaliterna M, Gronowicz G, Clark SH, Lichtler AC, Rowe D (2002) Use of type I collagen green fluorescent protein transgenes to identify subpopulations of cells at different stages of the osteoblast lineage. J Bone Miner Res 17:15–25

    Article  PubMed  CAS  Google Scholar 

  60. Malaval L, Liu F, Roche P, Aubin JE (1999) Kinetics of osteoprogenitor proliferation and osteoblast differentiation in vitro. J Cell Biochem 74:616–627

    Article  PubMed  CAS  Google Scholar 

  61. Krebsbach PH, Harrison JR, Lichtler AC, Woody CO, Rowe DW, Kream BE (1993) Transgenic expression of COL1A1-chloramphenicol acetyltransferase fusion genes in bone: differential utilization of promoter elements in vivo and in cultured cells. Mol Cell Biol 13:5168–5174

    PubMed  CAS  Google Scholar 

  62. Liu F, Malaval L, Gupta AK, Aubin JE (1994) Simultaneous detection of multiple bone-related mRNAs and protein expression during osteoblast differentiation: polymerase chain reaction and immunocytochemical studies at the single cell level. Dev Biol 166:220–234

    Article  PubMed  CAS  Google Scholar 

  63. Kalajzic Z, Liu P, Kalajzic I, Du Z, Braut A, Mina M, Canalis E, Rowe DW (2002) Directing the expression of a green fluorescent protein transgene in differentiated osteoblasts: comparison between rat type I collagen and rat osteocalcin promoters. Bone 31:654–660

    Article  PubMed  CAS  Google Scholar 

  64. Clover J, Dodds RA, Gowen M (1992) Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture. J Cell Sci 103:267–271

    PubMed  CAS  Google Scholar 

  65. Hughes DE, Salter DM, Dedhar S, Simpson R (1993) Integrin expression in human bone. J Bone Miner Res 8:527–533

    Article  PubMed  CAS  Google Scholar 

  66. Grzesik WJ, Robey PG (1994) Bone matrix RGD glycoproteins: immunolocalization and interaction with human primary osteoblastic bone cells in vitro. J Bone Miner Res 9:487–496

    PubMed  CAS  Google Scholar 

  67. Saito T, Albelda SM, Brighton CT (1994) Identification of integrin receptors on cultured human bone cells. J Orthop Res 12:384–394

    Article  PubMed  CAS  Google Scholar 

  68. Globus RK, Doty SB, Lull JC, Holmuhamedov E, Humphries MJ, Damsky CH (1998) Fibronectin is a survival factor for differentiated osteoblasts. J Cell Sci 111:1385–1393

    PubMed  CAS  Google Scholar 

  69. de la Fuente MT, Casanova B, Moyano JV, Garcia-Gila M, Sanz L, Garcia-Marco J, Silva A, Garcia-Pardo A (2002) Engagement of α4β1 integrin by fibronectin induces in vitro resistance of B chronic lymphocytic leukemia cells to fludarabine. J Leukoc Biol 71:495–502

    Google Scholar 

  70. Zhang Z, Vuori K, Reed JC, Ruoslahti E (1995) The α5β1 integrin supports survival of cells on fibronectin and up-regulates Bcl-2 expression. Proc Natl Acad Sci USA 92:6161–6165

    Article  PubMed  CAS  Google Scholar 

  71. Mak KK, Chen MH, Day TF, Chuang PT, Yang Y (2006) Wnt/β-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation. Development 133:3695–3707

    Article  PubMed  CAS  Google Scholar 

  72. Rodda SJ, McMahon AP (2006) Distinct roles for Hedgehog and canonical Wnt signaling in specification, differentiation and maintenance of osteoblast progenitors. Development 133:3231–3244

    Article  PubMed  CAS  Google Scholar 

  73. Gohel A, McCarthy MB, Gronowicz G (1999) Estrogen prevents glucocorticoid-induced apoptosis in osteoblasts in vivo and in vitro. Endocrinology 140:5339–5347

    Article  PubMed  CAS  Google Scholar 

  74. Giancotti FG, Ruoslahti E (1999) Integrin signaling. Science 285:1028–1032

    Article  PubMed  CAS  Google Scholar 

  75. Favus MJ (2006) Primer on the Metabolic Bone Diseases and Disorders of Mineral Metabolism. American Society for Bone and Mineral Research, Washington DC

    Google Scholar 

  76. Hock JM, Gunness-Hey M, Poser J, Olson H, Bell NH, Raisz LG (1986) Stimulation of undermineralized matrix formation by 1,25 dihydroxyvitamin D3 in long bones of rats. Calcif Tissue Int 38:79–86

    Article  PubMed  CAS  Google Scholar 

  77. Wronski TJ, Halloran BP, Bikle DD, Globus RK, Morey-Holton ER (1986) Chronic administration of 1,25-dihydroxyvitamin D3: increased bone but impaired mineralization. Endocrinology 119:2580–2585

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgment

We thank Dr. Mark Kronenberg (University of Connecticut Health Center) for providing mouse osterix cDNA. This work was supported by grant AR38933 from the National Institutes of Arthritis and Musculoskeletal Diseases (to G. G.).

Author information

Authors and Affiliations

  1. Department of Orthopedic Surgery, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT, 06030, USA

    W. Zhang, A. G. Pantschenko, M.-B. McCarthy & G. Gronowicz

Authors
  1. W. Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. G. Pantschenko
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M.-B. McCarthy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. G. Gronowicz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to G. Gronowicz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zhang, W., Pantschenko, A.G., McCarthy, MB. et al. Bone-Targeted Overexpression of Bcl-2 Increases Osteoblast Adhesion and Differentiation and Inhibits Mineralization In Vitro . Calcif Tissue Int 80, 111–122 (2007). https://doi.org/10.1007/s00223-006-0168-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00223-006-0168-2

Keywords

Search

Navigation