Cellular and Molecular Bioengineering

, Volume 8, Issue 1, pp 51–62 | Cite as

N-Cadherin-Mediated Signaling Regulates Cell Phenotype for Nucleus Pulposus Cells of the Intervertebral Disc

  • Priscilla Y. Hwang
  • Liufang Jing
  • Keith W. Michael
  • William J. Richardson
  • Jun Chen
  • Lori A. Setton
Article

Abstract

Juvenile nucleus pulposus (NP) cells of the intervertebral disc (IVD) are large, vacuolated cells that form cell clusters with strong cell–cell interactions. With maturation and aging, NP cells lose their ability to form these cell clusters, with aging-associated changes in NP cell phenotype, morphology, and proteoglycan synthesis that may contribute to IVD degeneration. Therefore, it is important to understand the mechanisms governing juvenile NP cell cluster behavior towards the goal of revealing factors that can promote juvenile, healthy NP cell phenotypes. N-cadherin has been identified as a cell–cell adhesion marker that is present in juvenile NP cells, but disappears with age. The goal of this study was to reveal the importance of N-cadherin in regulating cell–cell interactions in juvenile NP cell cluster formation and test for a regulatory role in maintaining a juvenile NP phenotype in vitro. Juvenile porcine IVD cells, of notochordal origin, were promoted to form cell clusters in vitro, and analyzed for preservation of the juvenile NP phenotype. Additionally, cadherin-blocking experiments were performed to prevent cluster formation in order to study the importance of cluster formation in NP cell signaling. Findings reveal N-cadherin-mediated cell–cell contacts promote cell clustering behavior and regulate NP cell matrix production and preservation of NP-specific markers. Inhibition of N-cadherin-mediated contacts resulted in loss of all features of the juvenile NP cell. These results establish a regulatory role for N-cadherin in juvenile NP cells, and suggest that preservation of the N-cadherin mediated cell–cell contact is important for preserving juvenile NP cell phenotype and morphology.

Keywords

N-cadherin Intervertebral disc Nucleus pulposus Cell–cell interactions 

References

  1. 1.
    Angst, B. D., C. Marcozzi, and A. I. Magee. The cadherin superfamily: diversity in form and function. J. Cell Sci. 114(Pt 4):629–641, 2001.Google Scholar
  2. 2.
    Antoniou, J., et al. The human lumbar intervertebral disc: evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J. Clin. Invest. 98(4):996–1003, 1996.CrossRefMathSciNetGoogle Scholar
  3. 3.
    Biyani, A., and G. B. Andersson. Low back pain: pathophysiology and management. J. Am. Acad. Orthop. Surg. 12(2):106–115, 2004.Google Scholar
  4. 4.
    Boos, N., et al. Classification of age-related changes in lumbar intervertebral discs: 2002 Volvo Award in basic science. Spine (Phila Pa 1976) 27(23):2631–2644, 2002.CrossRefGoogle Scholar
  5. 5.
    Buckwalter, J. A. Aging and degeneration of the human intervertebral disc. Spine (Phila Pa 1976) 20(11):1307–1314, 1995.Google Scholar
  6. 6.
    Callister Jr, W. D. Materials Science and Engineering an Introduction (6th ed.). New York: Wiley, 2003.Google Scholar
  7. 7.
    Cao, L., F. Guilak, and L. A. Setton. Three-dimensional morphology of the pericellular matrix of intervertebral disc cells in the rat. J. Anat. 211(4):444–452, 2007.Google Scholar
  8. 8.
    Charrasse, S., et al. N-cadherin-dependent cell-cell contact regulates Rho GTPases and beta-catenin localization in mouse C2C12 myoblasts. J. Cell Biol. 158(5):953–965, 2002.CrossRefGoogle Scholar
  9. 9.
    Chen, J., W. Yan, and L. A. Setton. Molecular phenotypes of notochordal cells purified from immature nucleus pulposus. Eur. Spine J. 15(Suppl 3):S303–S311, 2006.CrossRefGoogle Scholar
  10. 10.
    Chen, J., et al. Expression of laminin isoforms, receptors, and binding proteins unique to nucleus pulposus cells of immature intervertebral disc. Connect Tissue Res. 50(5):294–306, 2009.CrossRefGoogle Scholar
  11. 11.
    Cloyd, J. M., et al. Material properties in unconfined compression of human nucleus pulposus, injectable hyaluronic acid-based hydrogels and tissue engineering scaffolds. Eur. Spine J. 16(11):1892–1898, 2007.CrossRefGoogle Scholar
  12. 12.
    Delise, A. M., and R. S. Tuan. Analysis of N-cadherin function in limb mesenchymal chondrogenesis in vitro. Dev. Dyn. 225(2):195–204, 2002.CrossRefGoogle Scholar
  13. 13.
    Francisco, A. T., et al. Injectable laminin-functionalized hydrogel for nucleus pulposus regeneration. Biomaterials 34(30):7381–7388, 2013.CrossRefGoogle Scholar
  14. 14.
    Francisco, A. T., et al. Photocrosslinkable laminin-functionalized polyethylene glycol hydrogel for intervertebral disc regeneration. Acta Biomater. 10(3):1102–1111, 2014.CrossRefGoogle Scholar
  15. 15.
    Gilchrist, C. L., et al. Functional integrin subunits regulating cell-matrix interactions in the intervertebral disc. J. Orthop. Res. 25(6):829–840, 2007.CrossRefGoogle Scholar
  16. 16.
    Gilchrist, C. L., et al. Extracellular matrix ligand and stiffness modulate immature nucleus pulposus cell-cell interactions. PLoS One 6(11):e27170, 2011.CrossRefGoogle Scholar
  17. 17.
    Gilchrist, C. L., et al. Nucleus pulposus cell-matrix interactions with laminins. Eur. Cell Mater. 21:523–532, 2011.Google Scholar
  18. 18.
    Gruber, H. E., and E. N. Hanley, Jr. Human disc cells in monolayer vs 3D culture: cell shape, division and matrix formation. BMC Musculoskelet. Disord. 1:1, 2000.CrossRefGoogle Scholar
  19. 19.
    Halbleib, J. M., and W. J. Nelson. Cadherins in development: cell adhesion, sorting, and tissue morphogenesis. Genes Dev. 20(23):3199–3214, 2006.CrossRefGoogle Scholar
  20. 20.
    Harris, T. J., and U. Tepass. Adherens junctions: from molecules to morphogenesis. Nat. Rev. Mol. Cell Biol. 11(7):502–514, 2010.CrossRefGoogle Scholar
  21. 21.
    Hastreiter, D., R. M. Ozuna, and M. Spector. Regional variations in certain cellular characteristics in human lumbar intervertebral discs, including the presence of α-smooth muscle actin. J. Orthop. Res. 19(4):597–604, 2001.CrossRefGoogle Scholar
  22. 22.
    Hayes, A. J., M. Benjamin, and J. R. Ralphs. Extracellular matrix in development of the intervertebral disc. Matrix Biol. 20(2):107–121, 2001.CrossRefGoogle Scholar
  23. 23.
    Heuberger, J., and W. Birchmeier. Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling. Cold Spring Harb. Perspect. Biol. 2(2):a002915, 2010.CrossRefGoogle Scholar
  24. 24.
    Hunter, C. J., J. R. Matyas, and N. A. Duncan. The functional significance of cell clusters in the notochordal nucleus pulposus: survival and signaling in the canine intervertebral disc. Spine (Phila Pa 1976) 29(10):1099–1104, 2004.CrossRefGoogle Scholar
  25. 25.
    Hunter, C. J., J. R. Matyas, and N. A. Duncan. Cytomorphology of notochordal and chondrocytic cells from the nucleus pulposus: a species comparison. J. Anat. 205(5):357–362, 2004.CrossRefGoogle Scholar
  26. 26.
    Hwang, P. Y., et al. The role of extracellular matrix elasticity and composition in regulating the nucleus pulposus cell phenotype in the intervertebral disc: a narrative review. J. Biomech. Eng. 136(2):021010, 2014.CrossRefGoogle Scholar
  27. 27.
    Iatridis, J. C., et al. Is the nucleus pulposus a solid or a fluid? Mechanical behaviors of the nucleus pulposus of the human intervertebral disc. Spine (Phila Pa 1976) 21(10):1174–1184, 1996.CrossRefGoogle Scholar
  28. 28.
    Kleinman, H. K., and G. R. Martin. Matrigel: basement membrane matrix with biological activity. Semin. Cancer Biol. 15(5):378–386, 2005.CrossRefGoogle Scholar
  29. 29.
    Leckband, D., and A. Prakasam. Mechanism and dynamics of cadherin adhesion. Annu. Rev. Biomed. Eng. 8:259–287, 2006.CrossRefGoogle Scholar
  30. 30.
    Liebscher, T., et al. Age-related variation in cell density of human lumbar intervertebral disc. Spine (Phila Pa 1976) 36(2):153–159, 2011.CrossRefGoogle Scholar
  31. 31.
    Ludwinski, F. E., et al. Understanding the native nucleus pulposus cell phenotype has important implications for intervertebral disc regeneration strategies. Regen. Med. 8(1):75–87, 2013.CrossRefGoogle Scholar
  32. 32.
    Lv, F., et al. In search of nucleus pulposus-specific molecular markers. Rheumatology 53:600–610, 2014.CrossRefGoogle Scholar
  33. 33.
    Minogue, B. M., et al. Characterization of the human nucleus pulposus cell phenotype and evaluation of novel marker gene expression to define adult stem cell differentiation. Arthritis Rheum. 62(12):3695–3705, 2010.CrossRefGoogle Scholar
  34. 34.
    Minogue, B. M., et al. Transcriptional profiling of bovine intervertebral disc cells: implications for identification of normal and degenerate human intervertebral disc cell phenotypes. Arthritis Res. Ther. 12(1):R22, 2010.CrossRefGoogle Scholar
  35. 35.
    Murray, C. J., et al. The state of US health, 1990-2010: burden of diseases, injuries, and risk factors. JAMA 310(6):591–608, 2013.CrossRefGoogle Scholar
  36. 36.
    Nathke, I. S., et al. Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells. J. Cell Biol. 125(6):1341–1352, 1994.CrossRefGoogle Scholar
  37. 37.
    Plopper, G. E., et al. Migration of breast epithelial cells on Laminin-5: differential role of integrins in normal and transformed cell types. Breast Cancer Res. Treat. 51(1):57–69, 1998.CrossRefGoogle Scholar
  38. 38.
    Rodrigues-Pinto, R., S. M. Richardson, and J. A. Hoyland. Identification of novel nucleus pulposus markers: interspecies variations and implications for cell-based therapies for intervertebral disc degeneration. Bone Joint Res. 2(8):169–178, 2013.CrossRefGoogle Scholar
  39. 39.
    Roughley, P. J. Biology of intervertebral disc aging and degeneration: involvement of the extracellular matrix. Spine (Phila Pa 1976) 29(23):2691–2699, 2004.CrossRefGoogle Scholar
  40. 40.
    Sakai, D. Future perspectives of cell-based therapy for intervertebral disc disease. Eur. Spine J. 17(Suppl 4):452–458, 2008.CrossRefGoogle Scholar
  41. 41.
    Simon, R., et al. Analysis of gene expression data using BRB-ArrayTools. Cancer Inform. 3:11–17, 2007.Google Scholar
  42. 42.
    Stepniak, E., G. L. Radice, and V. Vasioukhin. Adhesive and signaling functions of cadherins and catenins in vertebrate development. Cold Spring Harb. Perspect. Biol. 1(5):a002949, 2009.CrossRefGoogle Scholar
  43. 43.
    Tam, V., V. Leung, and K. M. C. Cheung. Biological treatment for intervertebral disc degeneration to preserve motion—reality or fantasy? Eur. Musculoskelet. Rev. 7(1):5, 2012.Google Scholar
  44. 44.
    Tang, X., L. Jing, and J. Chen. Changes in the molecular phenotype of nucleus pulposus cells with intervertebral disc aging. PLoS One 7(12):e52020, 2012.CrossRefGoogle Scholar
  45. 45.
    Urban, J. P., and S. Roberts. Degeneration of the intervertebral disc. Arthritis Res. Ther. 5(3):120–130, 2003.CrossRefGoogle Scholar
  46. 46.
    Van den Bossche, J., et al. Regulation and function of the E-cadherin/catenin complex in cells of the monocyte-macrophage lineage and DCs. Blood 119(7):1623–1633, 2012.CrossRefGoogle Scholar
  47. 47.
    Wang, Z., et al. E-cadherin upregulates expression of matrix macromolecules aggrecan and collagen II in the intervertebral disc cells through activation of the intracellular BMP-Smad1/5 pathway. J. Orthop. Res. 30(11):1746–1752, 2012.CrossRefGoogle Scholar
  48. 48.
    Wheelock, M. J., and K. R. Johnson. Cadherin-mediated cellular signaling. Curr. Opin. Cell Biol. 15(5):509–514, 2003.CrossRefGoogle Scholar
  49. 49.
    Wheelock, M. J., and K. R. Johnson. Cadherins as modulators of cellular phenotype. Annu. Rev. Cell Dev. Biol. 19:207–235, 2003.CrossRefGoogle Scholar
  50. 50.
    Yap, A. S., and E. M. Kovacs. Direct cadherin-activated cell signaling: a view from the plasma membrane. J. Cell Biol. 160(1):11–16, 2003.CrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2014

Authors and Affiliations

  • Priscilla Y. Hwang
    • 1
  • Liufang Jing
    • 1
  • Keith W. Michael
    • 3
  • William J. Richardson
    • 2
  • Jun Chen
    • 2
  • Lori A. Setton
    • 1
    • 2
  1. 1.Department of Biomedical EngineeringDuke UniversityDurhamUSA
  2. 2.Department of Orthopaedic SurgeryDuke University Medical CenterDurhamUSA
  3. 3.Department of OrthopaedicsEmory Spine CenterAtlantaUSA

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