Journal of Molecular Medicine

, Volume 96, Issue 3–4, pp 333–347 | Cite as

Notch expressed by osteocytes plays a critical role in mineralisation

  • Jin Shao
  • Yinghong Zhou
  • Jinying Lin
  • Trung Dung Nguyen
  • Rong Huang
  • Yuantong Gu
  • Thor Friis
  • Ross Crawford
  • Yin XiaoEmail author
Original Article


Notch is actively involved in various life processes including osteogenesis; however, the role of Notch signalling in the terminal mineralisation of bone is largely unknown. In this study, it was noted that Hey1, a downstream target of Notch signalling was highly expressed in mature osteocytes compared to osteoblasts, indicating a potential role of Notch in osteocytes. Using a recently developed thermosensitive cell line (IDG-SW3), we demonstrated that dentin matrix acidic phosphoprotein 1 (DMP1) expression was inhibited and mineralisation process was significantly altered when Notch pathway was inactivated via administration of N-[N-(3,5-Difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), an inhibitor of Notch. Dysregulation of Notch in osteocyte differentiation can result in spontaneous deposition of calcium phosphate on collagen fibrils, disturbed transportation of intracellular mineral vesicles, alteration of mineral crystal structure, decreased bonding force between minerals and organic matrix, and suppression of dendrite development coupled with decreased expression of E11. In conclusion, the evidence presented here suggests that Notch plays a critical role in osteocyte differentiation and biomineralisation process.

Key messages

  • Notch plays a regulatory role in osteocyte phenotype.

  • Notch modulates the mineralisation mediated by osteocytes.

  • Notch activity influences the ultrastructural properties of bone mineralisation.


Notch Mineralisation Osteocytes DMP1 Osteoblasts 



This study was supported by the National Health and Medical Research Council (NHMRC) of Australia Early Career Fellowship (1105035) to YZ, QUT Tuition Waiver Scholarship to JS, and Science and Technology Project Grants funded by Xiamen Science and Technology Bureau (3502Z20161247) to JL, YZ and YX. The authors acknowledge the facilities, and the scientific and technical assistance of Dr. Jamie Riches, Ms. Rachel Hancock and Ms. Ning Liu, of the Australian Microscopy & Microanalysis Research Facility at the Central Analytical Research Facility operated by the Institute for Future Environments at the Queensland University of Technology.

Compliance with ethical standards

The study is approved by the Animal Ethics Committee of Queensland University of Technology

Supplementary material

109_2018_1625_MOESM1_ESM.docx (1.1 mb)
ESM 1 (DOCX 1170 kb)


  1. 1.
    Clarke B (2008) Normal bone anatomy and physiology. Clin J Am Soc Nephrol 3:S131–S139CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Canalis E, Giustina A, Bilezikian JP (2007) Mechanisms of anabolic therapies for osteoporosis. N Engl J Med 357:905–916CrossRefPubMedGoogle Scholar
  3. 3.
    Boonrungsiman S, Gentleman E, Carzaniga R, Evans ND, McComb DW, Porter AE, Stevens MM (2012) The role of intracellular calcium phosphate in osteoblast-mediated bone apatite formation. Proc Natl Acad Sci 109:14170–14175CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Mahamid J, Sharir A, Addadi L, Weiner S (2008) Amorphous calcium phosphate is a major component of the forming fin bones of zebrafish: indications for an amorphous precursor phase. Proc Natl Acad Sci 105:12748–12753CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Mahamid J, Sharir A, Gur D, Zelzer E, Addadi L, Weiner S (2011) Bone mineralization proceeds through intracellular calcium phosphate loaded vesicles: a cryo-electron microscopy study. J Struct Biol 174:527–535CrossRefPubMedGoogle Scholar
  6. 6.
    Toyosawa S, Shintani S, Fujiwara T, Ooshima T, Sato A, Ijuhin N, Komori T (2001) Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts. J Bone Miner Res 16:2017–2026CrossRefPubMedGoogle Scholar
  7. 7.
    Deshpande AS, Fang P-A, Zhang X, Jayaraman T, Sfeir C, Beniash E (2011) Primary structure and phosphorylation of dentin matrix protein 1 (DMP1) and dentin phosphophoryn (DPP) uniquely determine their role in biomineralization. Biomacromolecules 12:2933–2945CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Feng JQ, Ward LM, Liu S, Lu Y, Xie Y, Yuan B, Yu X, Rauch F, Davis SI, Zhang S, Rios H, Drezner MK, Quarles LD, Bonewald LF, White KE (2006) Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38:1310–1315CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Lu Y, Yuan B, Qin C, Cao Z, Xie Y, Dallas SL, McKee MD, Drezner MK, Bonewald LF, Feng JQ (2011) The biological function of DMP-1 in osteocyte maturation is mediated by its 57-kDa C-terminal fragment. J Bone Miner Res 26:331–340CrossRefPubMedGoogle Scholar
  10. 10.
    Orimo H (2010) The mechanism of mineralization and the role of alkaline phosphatase in health and disease. Journal of Nippon Medical School=Nippon Ika Daigaku zasshi 77:4–12CrossRefPubMedGoogle Scholar
  11. 11.
    Zhang K, Barragan-Adjemian C, Ye L, Kotha S, Dallas M, Lu Y, Zhao S, Harris M, Harris SE, Feng JQ, Bonewald LF (2006) E11/gp38 selective expression in osteocytes: regulation by mechanical strain and role in dendrite elongation. Mol Cell Biol 26:4539–4552CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Schulze E, Witt M, Kasper M, Lowik CW, Funk RH (1999) Immunohistochemical investigations on the differentiation marker protein E11 in rat calvaria, calvaria cell culture and the osteoblastic cell line ROS 17/2.8. Histochem Cell Biol 111:61–69CrossRefPubMedGoogle Scholar
  13. 13.
    Matsui K, Breiteneder-Geleff S, Kerjaschki D (1998) Epitope-specific antibodies to the 43-kD glomerular membrane protein podoplanin cause proteinuria and rapid flattening of podocytes. Journal of the American Society of Nephrology: JASN 9:2013–2026PubMedGoogle Scholar
  14. 14.
    Scholl FG, Gamallo C, Vilaro S, Quintanilla M (1999) Identification of PA2.26 antigen as a novel cell-surface mucin-type glycoprotein that induces plasma membrane extensions and increased motility in keratinocytes. J Cell Sci 112(Pt 24):4601–4613PubMedGoogle Scholar
  15. 15.
    Burra S, Nicolella DP, Francis WL, Freitas CJ, Mueschke NJ, Poole K, Jiang JX (2010) Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels. Proc Natl Acad Sci 107:13648–13653CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Piemontese M, Onal M, Xiong J, Han L, Thostenson JD, Almeida M, O’Brien CA (2016) Low bone mass and changes in the osteocyte network in mice lacking autophagy in the osteoblast lineage. Sci Rep 6:24262 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Thompson WR, Uzer G, Brobst KE, Xie Z, Sen B, Yen SS, Styner M, Rubin J (2015) Osteocyte specific responses to soluble and mechanical stimuli in a stem cell derived culture model. Sci Rep 5:11049 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Hesse B, Varga P, Langer M, Pacureanu A, Schrof S, Mannicke N, Suhonen H, Maurer P, Cloetens P, Peyrin F et al (2015) Canalicular network morphology is the major determinant of the spatial distribution of mass density in human bone tissue: evidence by means of synchrotron radiation phase-contrast nano-CT. J Bone and Mineral Res:Off J Am Soc Bone Mineral Res 30:346–356CrossRefGoogle Scholar
  19. 19.
    Nango N, Kubota S, Hasegawa T, Yashiro W, Momose A, Matsuo K (2016) Osteocyte-directed bone demineralization along canaliculi. Bone 84:279–288CrossRefPubMedGoogle Scholar
  20. 20.
    Turner CH, Robling AG, Duncan RL, Burr DB (2002) Do bone cells behave like a neuronal network? Calcif Tissue Int 70:435–442CrossRefPubMedGoogle Scholar
  21. 21.
    Buenzli PR, Sims NA (2015) Quantifying the osteocyte network in the human skeleton. Bone 75:144–150CrossRefPubMedGoogle Scholar
  22. 22.
    Schaffler MB, Cheung WY, Majeska R, Kennedy O (2014) Osteocytes: master orchestrators of bone. Calcif Tissue Int 94:5–24CrossRefPubMedGoogle Scholar
  23. 23.
    Genetos DC, Kephart CJ, Zhang Y, Yellowley CE, Donahue HJ (2007) Oscillating fluid flow activation of gap junction hemichannels induces atp release from MLO-Y4 osteocytes. J Cell Physiol 212:207–214CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Cherian PP, Siller-Jackson AJ, Gu S, Wang X, Bonewald LF, Sprague E, Jiang JX (2005) Mechanical strain opens connexin 43 hemichannels in osteocytes: a novel mechanism for the release of prostaglandin. Mol Biol Cell 16:3100–3106CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Andersson ER, Sandberg R, Lendahl U (2011) Notch signaling: simplicity in design, versatility in function. Development 138:3593–3612CrossRefPubMedGoogle Scholar
  26. 26.
    Nam Y, Sliz P, Song L, Aster JC, Blacklow SC (2006) Structural basis for cooperativity in recruitment of MAML coactivators to Notch transcription complexes. Cell 124:973–983CrossRefPubMedGoogle Scholar
  27. 27.
    Wilson JJ, Kovall RA (2006) Crystal structure of the CSL-Notch-mastermind ternary complex bound to DNA. Cell 124:985–996CrossRefPubMedGoogle Scholar
  28. 28.
    Kopan R, Ilagan MXG (2009) The canonical Notch signaling pathway: unfolding the activation mechanism. Cell 137:216–233CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Bray SJ (2016) Notch signalling in context. Nat Rev Mol Cell Biol. advance online publication 17:722–735CrossRefPubMedGoogle Scholar
  30. 30.
    Veno PND, Sivakumar P, Kalajzic I, Rowe D, Harris SE, Bonewald L, Dallas SL (2006) Live imaging of osteocytes within their lacunae reveals cell body and dendrite motions. J Bone Miner Res 21Google Scholar
  31. 31.
    Sprinzak D, Lakhanpal A, LeBon L, Santat LA, Fontes ME, Anderson GA, Garcia-Ojalvo J, Elowitz MB (2010) Cis-interactions between Notch and Delta generate mutually exclusive signalling states. Nature 465:86–90 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Rios AC, Serralbo O, Salgado D, Marcelle C (2011) Neural crest regulates myogenesis through the transient activation of NOTCH. Nature 473:532–535 CrossRefPubMedGoogle Scholar
  33. 33.
    Zhou Y, Fan W, Prasadam I, Crawford R, Xiao Y (2015) Implantation of osteogenic differentiated donor mesenchymal stem cells causes recruitment of host cells. J Tissue Eng Regen Med 9:118–126CrossRefPubMedGoogle Scholar
  34. 34.
    Ren Y, Lin S, Jing Y, Dechow PC, Feng JQ (2014) A novel way to statistically analyze morphologic changes in Dmp1-null osteocytes. Connect Tissue Res 55:129–133CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Shi M, Zhou Y, Shao J, Chen Z, Song B, Chang J, Wu C, Xiao Y (2015) Stimulation of osteogenesis and angiogenesis of hBMSCs by delivering Si ions and functional drug from mesoporous silica nanospheres. Acta Biomater 21:178–189CrossRefPubMedGoogle Scholar
  36. 36.
    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 17:15–25CrossRefPubMedGoogle Scholar
  37. 37.
    Li X, Liu P, Liu W, Maye P, Zhang J, Zhang Y, Hurley M, Guo C, Boskey A, Sun L, Harris SE, Rowe DW, Ke HZ, Wu D (2005) Dkk2 has a role in terminal osteoblast differentiation and mineralized matrix formation. Nat Genet 37:945–952 CrossRefPubMedGoogle Scholar
  38. 38.
    Woo SM, Rosser J, Dusevich V, Kalajzic I, Bonewald LF (2011) Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo. J Bone Miner Res 26:2634–2646CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Shih Y-RV, Hwang Y, Phadke A, Kang H, Hwang NS, Caro EJ, Nguyen S, Siu M, Theodorakis EA, Gianneschi NC, Vecchio KS, Chien S, Lee OK, Varghese S (2014) Calcium phosphate-bearing matrices induce osteogenic differentiation of stem cells through adenosine signaling. Proc Natl Acad Sci 111:990–995CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Li S, Shao J, Zhou Y, Friis T, Yao J, Shi B, Xiao Y (2016) The impact of Wnt signalling and hypoxia on osteogenic and cementogenic differentiation in human periodontal ligament cells. Mol Med Rep 14:4975–4982CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Moran P, Coats B (2012) Biological sample preparation for SEM imaging of porcine retina. Microscopy Today 20:28–31CrossRefGoogle Scholar
  42. 42.
    Fischer ER, Hansen BT, Nair V, Hoyt FH, Dorward DW (2012) Scanning electron microscopy. Curr Protoc Microbiol Chapter 2: Unit2B.2. DOI
  43. 43.
    Keene DR, Tufa SF (2010) Transmission electron microscopy of cartilage and bone. Methods Cell Biol 96:443–473CrossRefPubMedGoogle Scholar
  44. 44.
    Williams DB, Carter CB (2009) Diffraction from crystals transmission electron microscopy: a textbook for materials science. Springer US, Boston, pp 257–269Google Scholar
  45. 45.
    Zuo Q, Lu S, Du Z, Friis T, Yao J, Crawford R, Prasadam I, Xiao Y (2016) Characterization of nano-structural and nano-mechanical properties of osteoarthritic subchondral bone. BMC Musculoskelet Disord 17:367CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Nguyen TD, Gu Y (2016) Investigation of cell-substrate adhesion properties of living chondrocyte by measuring adhesive shear force and detachment using AFM and inverse FEA. Sci Rep 6:38059 CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Mikuni-Takagaki Y, Kakai Y, Satoyoshi M, Kawano E, Suzuki Y, Kawase T, Saito S (1995) Matrix mineralization and the differentiation of osteocyte-like cells in culture. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 10:231–242CrossRefGoogle Scholar
  48. 48.
    Kerschnitzki M, Kollmannsberger P, Burghammer M, Duda GN, Weinkamer R, Wagermaier W, Fratzl P (2013) Architecture of the osteocyte network correlates with bone material quality. Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 28:1837–1845CrossRefGoogle Scholar
  49. 49.
    Bell LS, Kayser M, Jones C (2008) The mineralized osteocyte: a living fossil. Am J Phys Anthropol 137:449–456CrossRefPubMedGoogle Scholar
  50. 50.
    Bonewald LF (2011) The amazing osteocyte. J Bone Miner Res 26:229–238CrossRefPubMedGoogle Scholar
  51. 51.
    Zanotti S, Canalis E (2010) Notch and the skeleton. Mol Cell Biol 30:886–896CrossRefPubMedGoogle Scholar
  52. 52.
    Zanotti S, Canalis E (2011) Notch regulation of bone development and remodeling and related skeletal disorders. Calcif Tissue Int 90:69–75CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Engin F, Yao Z, Yang T, Zhou G, Bertin T, Jiang MM, Chen Y, Wang L, Zheng H, Sutton RE, Boyce BF, Lee B (2008) Dimorphic effects of Notch signaling in bone homeostasis. Nat Med 14:299–305CrossRefPubMedPubMedCentralGoogle Scholar
  54. 54.
    Hilton MJ, Tu X, Wu X, Bai S, Zhao H, Kobayashi T, Kronenberg HM, Teitelbaum SL, Ross FP, Kopan R, Long F (2008) Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation. Nat Med 14:306–314CrossRefPubMedPubMedCentralGoogle Scholar
  55. 55.
    Dallas SL, Prideaux M, Bonewald LF (2013) The osteocyte: an endocrine cell…and more. Endocr Rev 34:658–690CrossRefPubMedPubMedCentralGoogle Scholar
  56. 56.
    Canalis E, Parker K, Feng JQ, Zanotti S (2013) Osteoblast lineage-specific effects of Notch activation in the skeleton. Endocrinology 154:623–634CrossRefPubMedGoogle Scholar
  57. 57.
    Canalis E, Bridgewater D, Schilling L, Zanotti S (2016) Canonical Notch activation in osteocytes causes osteopetrosis. Am J Physiol Endocrinol Metab 310:E171–E182CrossRefPubMedGoogle Scholar
  58. 58.
    Zanotti S, Smerdel-Ramoya A, Stadmeyer L, Durant D, Radtke F, Canalis E (2008) Notch inhibits osteoblast differentiation and causes osteopenia. Endocrinology 149:3890–3899CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Deregowski V, Gazzerro E, Priest L, Rydziel S, Canalis E (2006) Notch 1 overexpression inhibits osteoblastogenesis by suppressing Wnt/β-catenin but not bone morphogenetic protein signaling. J Biol Chem 281:6203–6210CrossRefPubMedGoogle Scholar
  60. 60.
    John HCS, Bishop KA, Meyer MB, Benkusky NA, Leng N, Kendziorski C, Bonewald LF, Pike JW (2014) The osteoblast to osteocyte transition: epigenetic changes and response to the vitamin D3 hormone. Mol Endocrinol 28:1150–1165CrossRefGoogle Scholar
  61. 61.
    Liu P, Ping Y, Ma M, Zhang D, Liu C, Zaidi S, Gao S, Ji Y, Lou F, Yu F, Lu P, Stachnik A, Bai M, Wei C, Zhang L, Wang K, Chen R, New MI, Rowe DW, Yuen T, Sun L, Zaidi M (2016) Anabolic actions of Notch on mature bone. Proc Natl Acad Sci 113:E2152–E2161CrossRefPubMedPubMedCentralGoogle Scholar
  62. 62.
    Addison WN, Nelea V, Chicatun F, Chien YC, Tran-Khanh N, Buschmann MD, Nazhat SN, Kaartinen MT, Vali H, Tecklenburg MM, Franceschi RT, McKee MD (2015) Extracellular matrix mineralization in murine MC3T3-E1 osteoblast cultures: an ultrastructural, compositional and comparative analysis with mouse bone. Bone 71:244–256CrossRefPubMedGoogle Scholar
  63. 63.
    Nudelman F, Pieterse K, George A, Bomans PHH, Friedrich H, Brylka LJ, Hilbers PAJ, de With G, Sommerdijk NAJM (2010) The role of collagen in bone apatite formation in the presence of hydroxyapatite nucleation inhibitors. Nat Mater 9:1004–1009 CrossRefPubMedPubMedCentralGoogle Scholar
  64. 64.
    He G, Dahl T, Veis A, George A (2003) Nucleation of apatite crystals in vitro by self-assembled dentin matrix protein 1. Nat Mater 2:552–558 CrossRefPubMedGoogle Scholar
  65. 65.
    He G, George A (2004) Dentin matrix protein 1 immobilized on type I collagen fibrils facilitates apatite deposition in vitro. J Biol Chem 279:11649–11656CrossRefPubMedGoogle Scholar
  66. 66.
    He G, Gajjeraman S, Schultz D, Cookson D, Qin C, Butler WT, Hao J, George A (2005) Spatially and temporally controlled biomineralization is facilitated by interaction between self-assembled dentin matrix protein 1 and calcium phosphate nuclei in solution. Biochemistry 44:16140–16148CrossRefPubMedPubMedCentralGoogle Scholar
  67. 67.
    Dey A, Bomans PHH, Müller FA, Will J, Frederik PM, de With G, Sommerdijk NAJM (2010) The role of prenucleation clusters in surface-induced calcium phosphate crystallization. Nat Mater 9:1010–1014 CrossRefPubMedGoogle Scholar
  68. 68.
    Narayanan K, Ramachandran A, Hao J, He G, Park KW, Cho M, George A (2003) Dual functional roles of dentin matrix protein 1: implications in biomineralization and gene transcription by activation of intracellular Ca2+ store. J Biol Chem 278:17500–17508CrossRefPubMedGoogle Scholar
  69. 69.
    Harris SE, Gluhak-Heinrich J, Harris MA, Yang W, Bonewald LF, Riha D, Rowe PSN, Robling AG, Turner CH, Feng JQ, McKee M, Nicollela D (2007) DMP1 and MEPE expression are elevated in osteocytes after mechanical loading in vivo: theoretical role in controlling mineral quality in the perilacunar matrix. J Musculoskelet Neuronal Interact 7:313–315PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Jin Shao
    • 1
    • 2
  • Yinghong Zhou
    • 1
    • 2
  • Jinying Lin
    • 2
    • 3
  • Trung Dung Nguyen
    • 4
    • 5
  • Rong Huang
    • 1
    • 2
  • Yuantong Gu
    • 2
    • 4
  • Thor Friis
    • 1
    • 2
  • Ross Crawford
    • 1
    • 2
    • 6
  • Yin Xiao
    • 1
    • 2
    Email author
  1. 1.Institute of Health and Biomedical InnovationQueensland University of TechnologyBrisbaneAustralia
  2. 2.The Australia–China Centre for Tissue Engineering and Regenerative Medicine (ACCTERM)Queensland University of TechnologyBrisbaneAustralia
  3. 3.Department of Implantology, Xiamen Stomatological Research InstituteXiamen Stomatological HospitalFujianChina
  4. 4.School of Chemistry, Physics and Mechanical Engineering, Science and Engineering FacultyQueensland University of TechnologyBrisbaneAustralia
  5. 5.Department of Aerospace and Mechanical Engineering, College of EngineeringUniversity of Notre DameNotre DameUSA
  6. 6.The Prince Charles HospitalBrisbaneAustralia

Personalised recommendations