Calcified Tissue International

, Volume 74, Issue 5, pp 448–457 | Cite as

Uni-axial Cyclic Stretch Induces Cbfa1 Expression in Spinal Ligament Cells Derived from Patients with Ossification of the Posterior Longitudinal Ligament

  • K. Iwasaki
  • K.-I. FurukawaEmail author
  • M. Tanno
  • T. Kusumi
  • K. Ueyama
  • M. Tanaka
  • H. Kudo
  • S. Toh
  • S. Harata
  • S. Motomura


Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. Mechanical stress, which acts on the posterior ligaments, is thought to be an important factor in the progression of OPLL. To clarify this mechanism, we investigated the effects of in vitro cyclic stretch (120% peak to peak, at 0.5 Hz) on cultured spinal ligament cells derived from OPLL (OPLL cells) and non-OPLL (non-OPLL cells) patients. The mRNA expressions of Cbfa1 (an osteoblast-specific transcription factor), type I collagen, alkaline phosphatase (ALP), osteocalcin and integrin β1 (a mechanotransducer) were increased by cyclic stretch in OPLL cells, whereas no change was observed in non-OPLL cells. The effects of cyclic stretch on the spinal ligament tissues derived from OPLL and non-OPLL patients were also analyzed by immunohistochemistry using an antibody against Cbfa1. The expression of Cbfa1 was increased by cyclic stretch at the center of the spinal ligament tissues of OPLL patients, whereas no change was observed in the tissues of non-OPLL patients. Furthermore, U0126, a specific inhibitor of MAPK kinase (MEK), suppressed the stretch-induced mRNA expressions of Cbfa1, ALP and type I collagen in OPLL cells. These results suggest that in OPLL cells, mechanical stress is converted by integrin β1 into intracellular signaling and that Cbfa1 is activated through the MAP kinase pathway. Therefore, we propose that mechanical stress plays a key role in the progression of OPLL through an increase in Cbfa1 expression.


Posterior longitudinal ligament Core-binding factor alpha 1 Integrin β1 Mechanical stress Ossification Immunohistochemistry 



We thank Drs. Kazuhiko Seya and Miki Hashimoto of the Department of Pharmacology, Hirosaki University School of Medicine and Drs. Hirotaka Ohishi and Tomohiro Iwasawa of the Department of Orthopaedic Surgery, for their technical assistance. We also acknowledge professors Hiroto Kimura, Keiichi Takagaki and Hideki Mizunuma, Hirosaki University School of Medicine, for their valuable suggestions.


  1. 1.
    Resnick, D, Shaul, SR, Robins, JM 1975Diffuse idiopathic skeletal hyperostosis (DISH).Radiography115513524Google Scholar
  2. 2.
    Wang, PN, Chen, SS, Liu, HC, Fuh, JL, Kuo, BI, Wang, SJ 1999Ossification of the posterior longitudinal ligament of the spine. A case-control risk factor study.Spine24142144CrossRefPubMedGoogle Scholar
  3. 3.
    Baba, H, Furusawa, N, Fukuda, M, Maezawa, Y, Imura, S, Kawahara, N, Nakahashi, K, Tomita, K 1997Potential role of streptozotocin in enhancing ossification of the posterior longitudinal ligament of the cervical spine in hereditary spinal hyperostotic mouse (twy/twy).Eur J Histochem41191202PubMedGoogle Scholar
  4. 4.
    Ishida, Y, Kawai, S 1993Characteraixation of cultured cells derived from ossification of the posterior longitudinal ligament of the spine.Bone148591PubMedGoogle Scholar
  5. 5.
    Inaba, K, Matsunaga, S, Ishidou, Y, Imamura, T, Yoshida, H 1996Effect of transforming growth factor-beta on fibroblasts in ossification of the posterior longitudinal ligament.In Vivo10445449PubMedGoogle Scholar
  6. 6.
    Kon, T, Yamazaki, M, Tagawa, M, Goto, S, Terakado, A, Moriya, H, Fujimura, S 1997Bone morphogenetic protein-2 stimulates differentiation of cultured spnal ligament cells from patients with ossification of the posterior longitudinal ligament.Calcif Tissue Int60291296CrossRefPubMedGoogle Scholar
  7. 7.
    Goto, K, Yamazaki, M, Tagawa, M, Goto, S, Kon, T, Moriya, H, Fujimura, S 1998Involvement of insuline-like growth factor I in development of ossification the posterior longitudinal ligament of the spine.Calcif Tissue Int62158165Google Scholar
  8. 8.
    Ishida, Y, Kawai, S 1993Effects of bone-seeking hormones on DNA synthesis, cyclic AMP level, and alkaline phosphatase activity in cultured cells from human posterior longitudinal ligament of the spine.J Bone Miner Res812911300Google Scholar
  9. 9.
    Yonemori, K, Imamura, T, Ishidou, Y, Okano, T, Matsunaga, S, Yoshida, H, Kato, M, Sampath, TK, Miyazono, K, ten Dijke, P, Sakou, T 1997Bone morphogenetic protein receptors and activin receptors are highly expressed in ossified ligament tissues of patients with ossification of the posterior longitudinal ligament.Am J Pathol15013351347PubMedGoogle Scholar
  10. 10.
    Koga, H, Sakou, T, Taketomi, E, Hayashi, K, Numasawa, T, Harata, S, Yone, K, Matsunaga, S, Otterud, B, Inoue, I, Leppert, M 1998Genetic mapping of ossification of the posterior longitudinal ligament of the spine.Am J Hum Genet6214601467Google Scholar
  11. 11.
    Numasawa, T, Koga, H, Ueyama, K, Maeda, S, Sakou, T, Harata, S, Leppert, M, Inoue, I 1999Human retinoic X receptor beta: complete genomic sequence and mutation search for ossification of posterior longitudinal ligament of the spine.J Bone Miner Res14500508Google Scholar
  12. 12.
    Furushima, K, Shimo-onoda, K, Maeda, S, Nobukuni, T, Ikari, K, Koga, H, Komiya, S, Nakajima, T, Harata, S, Inoue, I 2002Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine.J Bone Miner Res17128137Google Scholar
  13. 13.
    Matsunaga, S, Sakou, T, Taketomi, E, Yamaguchi, M, Okano, T 1994The natural course of myelopathy caused by ossification of the posterior longitudinal ligament in the cervical spine.Clin. Orthop305168177PubMedGoogle Scholar
  14. 14.
    Nakamura, H 1994A radiographic study of the progression of ossification of the cervical posterior longitudinal ligament: the correlation between the ossification of the posterior longitudinal ligament and that of the anterior longitudinal ligament.Nippon Seikeigeka Gakkai Zasshi68725736PubMedGoogle Scholar
  15. 15.
    Takatsu, T, Ishida, Y, Suzuki, K, Inoue, H 1999Radiological study of cervical ossification of the posterior longitudinal ligament.J Spinal Disord12271273PubMedGoogle Scholar
  16. 16.
    Matsunaga, S, Sakou, T, Taketomi, E, Nakanishi, K 1996Effects of strain distribution in the intervertebral discs on the progression of ossification of the posterior longitudinal ligaments.Spine21184189CrossRefPubMedGoogle Scholar
  17. 17.
    Yamamoto, Y, Furukawa, K, Ueyama, K, Nakanishi, T, Takigawa, M, Harata, S 2002Possible roles of CTGF/Hcs24 in the initiation and development of ossification of the posterior longitudinal ligament.Spine2718521857CrossRefPubMedGoogle Scholar
  18. 18.
    Karsenty, G 1999The genetic transformation of bone biology.Genes Dev1330373051CrossRefPubMedGoogle Scholar
  19. 19.
    Ducy, P, Karsenty, G 1995Two distinct osteoblastic-specific cis-acting elements control expression of a mouse osteocalcin gene.Mol Cell Biol1518581869PubMedGoogle Scholar
  20. 20.
    Harada, H, Tagashira, S, Fujiwara, M, Ogawa, S, Katsumata, T, Yamaguchi, A, Komori, T, Nakatsuka, M 1999Cbfa1 isoforms exert functional differences in osteoblast differentiation.J Biol Chem274697269678PubMedGoogle Scholar
  21. 21.
    Kern, B, Shen, J, Starbuck, M, Karsenty, G 2001Cbfa1 contributes to the osteoblast-specific expression of Type I collagen genes.J Biol Chem27671017107CrossRefPubMedGoogle Scholar
  22. 22.
    Ducy, P, Zhang, R, Geoffroy, V, Ridall, AL, Karsenty, G 1997Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation.Cell89747774PubMedGoogle Scholar
  23. 23.
    Ducy, P, Starbuck, M, Priemel, M, Shen, J, Pinero, G, Geoffrey, V, Amling, M, Karsenty, G 1999A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development.Genes Dev1310251036PubMedGoogle Scholar
  24. 24.
    Hipskind, RA, Bilbe, G 1998MAP kinase signaling cascades and gene expression in osteoblasts.Front Biosci3D804D816PubMedGoogle Scholar
  25. 25.
    Lou, J, Tu, Y, Li, S, Manske, PR 2000Involvement of ERK in BMP-2 induced osteoblastic differentiation of mesenchymal progenitor cell line C3H10T1/2.Biochem Biophys Res Commun268757762CrossRefPubMedGoogle Scholar
  26. 26.
    Gallea, S, Lallemand, F, Atfi, A, Rawadi, G, Ramez, V, Spinella-Jaegle, S, Kawai, S, Faucheu, C, Huet, L, Baron, R, Roman-Roman, S 2001Activation of mitogen-activated protein kinase cascades is involved in regulation of bone morphogenetic protein-2-induced osteoblast differentiation in pluripotent C2C12 cells.Bone28491498CrossRefPubMedGoogle Scholar
  27. 27.
    Huang, Z, Cheng, SL, Slatopolsky, E 2001Sustained activation of the extracellular signal-regulated kinase pathway is required for extracellular calcium stimulation of human osteoblast proliferation.J Biol Chem2762135121358CrossRefPubMedGoogle Scholar
  28. 28.
    Lai, CF, Chaudhary, L, Fausto, A, Halstead, LR, Ory, DS, Avioli, LV, Cheng, SL 2001Erk is essential for growth, differentiation, integral expression, and cell function in human osteoblastic cells.J Biol Chem2761444314450PubMedGoogle Scholar
  29. 29.
    Ingber, D 1991Integrins as mechanochemical transducers.Curr Opin Cell Biol3841848PubMedGoogle Scholar
  30. 30.
    MacKenna, DA, Dolfi, F, Vuori, K, Ruoslahti, E 1998Extracellular signal-regulated kinase and c-Jun NH2-terminal kinase activation by mechanical stretch is integrin-dependent and matrix-specific in rat cardiac fibroblasts.J Clin Invest101301310PubMedGoogle Scholar
  31. 31.
    Pavalko, FM, Chen, NX, Turner, CH, Burr, DB, Atkinson, S, Hsieh, YF, Qiu, J, Duncan, RL 1998Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions.Am J Physiol275C15911601PubMedGoogle Scholar
  32. 32.
    Schmidt, C, Pommerenke, H, Durr, F, Nebe, B, Rychly, J 1998Mechanical stressing of integrin receptors induces enhanced tyrosine phosphorylation of cytoskeletally anchored proteins.J Biol Chem27350815085PubMedGoogle Scholar
  33. 33.
    Pommerenke, H, Schmidt, C, Durr, F, Nebe, B, Luthen, F, Muller, P, Rychly, J 2002The mode of mechanical integrin stressing controls intracellular signaling in osteoblasts.J Bone Miner17603611Google Scholar
  34. 34.
    Togari, A, Arai, M, Mizutani, S, Mizutani, S, Koshihara, Y, Nagatsu, T 1997Expression of mRNAs for neuropeptide receptors and beta-adrenergic receptors in human osteoblasts and human osteogenic sarcoma cells.Neurosci Lett233125128CrossRefPubMedGoogle Scholar
  35. 35.
    Tou, L, Quibria, N, Alexander, JM 2001Regulation of human cbfa1 gene transcription in osteoblasts by selective estrogen receptor modulators.Mol Cell Endocrinol1837179CrossRefPubMedGoogle Scholar
  36. 36.
    Buckley, MJ, Banes, AJ, Jordan, RD 1990The effects of mechanical strain on osteoblasts in vitro.J Oral Maxillofac Surg48276282PubMedGoogle Scholar
  37. 37.
    Harter, LV, Hruska, KA, Duncan, RL 1995Human osteoblast-like cells respond to mechanical strain with increased bone matrix protein production independent of hormonal regulation.Endocrinology136528535PubMedGoogle Scholar
  38. 38.
    Pavlin, D, Dove, SB, Zadro, R, Gluhak-Heinrich, J 2000Mechanical loading stimulates differentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes.Calcif Tissue Int67163172Google Scholar
  39. 39.
    Miyagawa J, Tanaka K, Ohkuma T (1983) The range of motion of cervical spine in ossification of the posterior longitudinal ligament. Proceeding of the investigation committee on ossification of the spinal ligament. Tokyo: Investigation committee on ossification of the spinal ligament:168–176 (In Japanese)Google Scholar
  40. 40.
    Tominaga S (1981) The relationship between dynamic cervical motion and symptoms on myelopathy due to OPLL in the cervical spine. Proceeding of the investigation committee on ossification of the spinal ligament. Tokyo: Investigation committee on ossification of the spinal ligament:136–142 (In Japanese)Google Scholar
  41. 41.
    Nakamura H, Okajima Y, Hasegawa K (1993) Analysis of the dynamic cervical motion. Proceeding of the investigation committee on ossification of the spinal ligament. Tokyo: Investigation committee on ossification of the spinal ligament:173–176 (In Japanese)Google Scholar
  42. 42.
    Ducy, P 2000Cbfa1: a molecular switch in osteoblast biology.Dev Dyn219461471CrossRefPubMedGoogle Scholar
  43. 43.
    Karsenty, G 2000Role of Cbfa1 in osteoblast differentiation and function.Semin Cell Dev Biol11343346Google Scholar
  44. 44.
    Inada, M, Yasui, T, Nomura, S, Miyake, S, Deguchi, K, Himeno, M, Sato, M, Yamagiwa, H, Kimura, T, Yasui, N, Ochi, T, Endo, N, Kitamura, Y, Kishimoto, T, Komori, T 1999Maturational disturbance of chondrocytes in Cbfa1-deficient mice.Dev Dyn214279290CrossRefPubMedGoogle Scholar
  45. 45.
    Breen, EC 2000Mechanical strain increases type I collagen expression in pulmonary fibroblasts in vitro.J Appl Physiol88203209PubMedGoogle Scholar
  46. 46.
    Ziros, PG, Gil, AP, Georgakopoulos, T, Habeos, I, Kletsas, D, Basdra, EK, Papavassiliou, AG 2002The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells.J Biol Chem2772393423941CrossRefPubMedGoogle Scholar
  47. 47.
    Xiao, G, Gopalakrishnan, R, Jiang, D, Reith, E, Benson, MD, Franceschi, RT 2002Bone morphogenetic proteins, extracellular matrix, and mitogen-activated protein kinase signaling pathways are required for osteoblast-specific gene expression and differentiation in MC3T3-E1 cells.J Bone Miner Res17101110Google Scholar
  48. 48.
    Carvalho, RS, Scott, JE, Yen, EH 1995The effects of mechanical stimulation on the distribution of beta 1 integrin and expression of beta 1-integrin mRNA in TE-85 human osteosarcoma cells.Arch Oral Biol40257264CrossRefPubMedGoogle Scholar
  49. 49.
    Carvalho, RS, Bumann, A, Schwarzer, C, Scott, E, Yen, EH 1996A molecular mechanism of integrin regulation from bone cells stimulated by orthodontic forces.Eur J Orthod18227235PubMedGoogle Scholar
  50. 50.
    Kramer, RH, Marks, N 1989Identification of integrin collagen receptors on human melanoma cells.J Biol Chem26446844688PubMedGoogle Scholar
  51. 51.
    Vihinen, P, Riikonen, T, Laine, A, Heino, J 1996Integrin alpha 2 beta 1 in tumorigenic human osteosarcoma cell lines regulates cell adhesion, migration, and invasion by interaction with type I collagen.Cell Growth Differ7439447PubMedGoogle Scholar
  52. 52.
    Takeuchi, Y, Nakayama, K, Matsumoto, T 1996Differentiation and cell surface expression of transforming growth factor-beta receptors are regulated by interaction with matrix collagen in murine osteoblastic cells.J Biol Chem27139383944CrossRefPubMedGoogle Scholar
  53. 53.
    Jikko, A, Harris, SE, Chen, D, Mendrick, DL, Damsky, CH 1999Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2.J Bone Miner Res1410751083Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • K. Iwasaki
    • 1
    • 2
  • K.-I. Furukawa
    • 1
    Email author
  • M. Tanno
    • 1
    • 2
  • T. Kusumi
    • 3
  • K. Ueyama
    • 4
  • M. Tanaka
    • 3
  • H. Kudo
    • 3
  • S. Toh
    • 2
  • S. Harata
    • 5
  • S. Motomura
    • 1
  1. 1.Department of PharmacologyHirosaki University School of Medicine, HirosakiJapan
  2. 2.Department of Orthopaedic SurgeryHirosaki University School of Medicine, HirosakiJapan
  3. 3.Department of PathologyHirosaki University School of Medicine, HirosakiJapan
  4. 4.Department of Orthopaedic SurgeryHirosaki Memorial Hospital, HirosakiJapan
  5. 5.Aomori Prefectural Central Hospital, AomoriJapan

Personalised recommendations