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Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity


We investigated the effects of Rho-associated kinase (ROCK) on migration and cytoskeletal organization in primary human osteoblasts and Saos-2 human osteosarcoma cells. Both cell types were exposed to two different ROCK inhibitors, Y-27632 and HA-1077. In the improved motility assay used in the present study, Y-27632 and HA-1077 significantly increased the migration of both osteoblasts and osteosarcoma cells on plastic in a dose-dependent and reversible manner. Fluorescent images showed that cells of both types cultured with Y-27632 or HA-1077 exhibited a stellate appearance, with poor assembly of stress fibers and focal contacts. Western blotting showed that ROCK inhibitors reduced myosin light chain (MLC) phosphorylation within 5 min without affecting overall myosin light-chain protein levels. Inhibition of ROCK activity is thought to enhance the migration of human osteoblasts through reorganization of the actin cytoskeleton and regulation of myosin activity. ROCK inhibitors may be potentially useful as anabolic agents to enhance the biocompatibility of bone and joint prostheses.



charge-coupled device


epidermal growth factor


ethylenediaminetetraacetic acid


focal adhesion kinase


fibroblast growth factor


glyceraldehyde 3-phosphate dehydrogenase


guanosine triphopsphate


myosin light chain


myosin light chain kinase


platelet-derived growth factor


phospharylation of myosin light chain


Rho-associated kinase


sodium dodecyl sulfate-polyacrylamide gel electrophoresis


tris-buffered saline Tween 20


  1. 1.

    Williams, D.F. On the mechanisms of biocompatibility. Biomaterials 29 (2008) 2941–2953.

  2. 2.

    Wu, J., Liu, Z.M., Zhao, X.H., Gao, Y., Hu, J. and Gao, B. Improved biological performance of microarc-oxidized low-modulus Ti-24Nb-4Zr-7.9Sn alloy. J. Biomed. Mater. Res. B Appl. Biomater. 92B (2009) 298–306.

  3. 3.

    Park, J.W., Kim, H.K., Kim, Y.J., Jang, J.H., Song, H. and Hanawa, T. Osteoblast response and osseointegration of a Ti-6Al-4V alloy implant incorporating strontium. Acta Biomater. 7 (2010) 2843–2851.

  4. 4.

    Alves, S.F. and Wassall, T. In vitro evaluation of osteoblastic cell adhesion on machined osseointegrated implants. Braz. Oral Res. 23 (2009) 131–136.

  5. 5.

    Gurkan, U.A., Cheng, X., Kishore, V., Uquillas, J.A. and Akkus, O. Comparison of morphology, orientation, and migration of tendon derived fibroblasts and bone marrow stromal cells on electrochemically aligned collagen constructs. J. Biomed. Mater. Res. A 94 (2010) 1070–1079.

  6. 6.

    Nakamura, M., Nagai, A., Tanaka, Y., Sekijima, Y. and Yamashita, K. Polarized hydroxyapatite promotes spread and motility of osteoblastic cells. J. Biomed. Mater. Res. A 92 (2009) 783–790.

  7. 7.

    Miranda, L., Carpentier, S., Platek, A., Hussain, N., Gueuning, M.A., Vertommen, D., Ozkan, Y., Sid, B., Hue, L., Courtoy, P.J., Rider, M.H. and Horman, S. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells. Biochem. Biophys. Res. Commun. 396 (2010) 656–661.

  8. 8.

    Martini, F.J. and Valdeolmillos, M. Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons. J. Neurosci. 30 (2010) 8660–8670.

  9. 9.

    Schwartz, M.A. and Horwitz, A.R. Integrating adhesion, protrusion, and contraction during cell migration. Cell 125 (2006) 1223–1225.

  10. 10.

    Kaibuchi, K., Kuroda, S. and Amano, M. Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu. Rev. Biochem. 68 (1999) 459–486.

  11. 11.

    Hall, A. Rho GTPases and the actin cytoskeleton. Science 279 (1998) 509–514.

  12. 12.

    Gallagher, P.J., Herring, B.P. and Stull, J.T. Myosin light chain kinases. J. Muscle Res. Cell Motil. 18 (1997) 1–16.

  13. 13.

    Tokuda, H., Takai, S., Matsushima-Nishiwaki, R., Hanai, Y., Adachi, S., Minamitani, C., Mizutani, J., Otsuka, T. and Kozawa, O. Function of Rhokinase in prostaglandin D2-induced interleukin-6 synthesis in osteoblasts. Prostaglandins Leukot. Essent. Fatty Acids 79 (2008) 41–46.

  14. 14.

    Harmey, D., Stenbeck, G., Nobes, C.D., Lax, A.J. and Grigoriadis, A.E. Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation. J. Bone Miner. Res. 19 (2004) 661–670.

  15. 15.

    Kazmers, N.H., Ma, S.A., Yoshida, T. and Stern, P.H. Rho GTPase signaling and PTH 3–34, but not PTH 1–34, maintain the actin cytoskeleton and antagonize bisphosphonate effects in mouse osteoblastic MC3T3-E1 cells. Bone 45 (2009) 52–60.

  16. 16.

    Loirand, G., Guerin, P. and Pacaud, P. Rho kinases in cardiovascular physiology and pathophysiology. Circ. Res. 98 (2006) 322–334.

  17. 17.

    Zohrabian, V.M., Forzani, B., Chau, Z., Murali, R. and Jhanwar-Uniyal, M. Rho/ROCK and MAPK signaling pathways are involved in glioblastoma cell migration and proliferation. Anticancer Res. 29 (2009) 119–123.

  18. 18.

    Hahmann, C. and Schroeter, T. Rho-kinase inhibitors as therapeutics: from pan inhibition to isoform selectivity. Cell. Mol. Life Sci. 67 (2010) 171–177.

  19. 19.

    Lingor, P., Teusch, N., Schwarz, K., Mueller, R., Mack, H., Bahr, M. and Mueller, B.K. Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo. J. Neurochem. 103 (2007) 181–189.

  20. 20.

    Ikebe, M. and Hartshorne, D.J. Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J. Biol. Chem. 260 (1985) 10027–10031.

  21. 21.

    Totsukawa, G., Yamakita, Y., Yamashiro, S., Hartshorne, D.J., Sasaki, Y. and Matsumura, F. Distinct roles of ROCK (Rho-kinase) and MLCK in spatial regulation of MLC phosphorylation for assembly of stress fibers and focal adhesions in 3T3 fibroblasts. J. Cell Biol. 150 (2000) 797–806.

  22. 22.

    Gao, S.Y., Li, C.Y., Chen, J., Pan, L., Saito, S., Terashita, T., Saito, K., Miyawaki, K., Shigemoto, K., Mominoki, K., Matsuda, S. and Kobayashi, N. Rho-ROCK signal pathway regulates microtubule-based process formation of cultured podocytes—inhibition of ROCK promoted process elongation. Nephron Exp. Nephrol. 97 (2004) e49–61.

  23. 23.

    Li, C.Y., Gao, S.Y., Terashita, T., Shimokawa, T., Kawahara, H., Matsuda, S. and Kobayashi, N. In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces. Cell Tissue Res. 324 (2006) 369–375.

  24. 24.

    Celotti, F., Colciago, A., Negri-Cesi, P., Pravettoni, A., Zaninetti, R. and Sacchi, M.C. Effect of platelet-rich plasma on migration and proliferation of SaOS-2 osteoblasts: role of platelet-derived growth factor and transforming growth factor-beta. Wound Repair Regen. 14 (2006) 195–202.

  25. 25.

    Tsiridis, E., Upadhyay, N. and Giannoudis, P. Molecular aspects of fracture healing: which are the important molecules? Injury 38Suppl 1 (2007) S11–25.

  26. 26.

    Klein, M.O., Reichert, C., Koch, D., Horn, S. and Al-Nawas, B. In vitro assessment of motility and proliferation of human osteogenic cells on different isolated extracellular matrix components compared with enamel matrix derivative by continuous single-cell observation. Clin. Oral Implants Res. 18 (2007) 40–45.

  27. 27.

    Jacobs, M., Hayakawa, K., Swenson, L., Bellon, S., Fleming, M., Taslimi, P. and Doran, J. The structure of dimeric ROCK I reveals the mechanism for ligand selectivity. J. Biol. Chem. 281 (2006) 260–268.

  28. 28.

    Tamura, M., Nakao, H., Yoshizaki, H., Shiratsuchi, M., Shigyo, H., Yamada, H., Ozawa, T., Totsuka, J. and Hidaka, H. Development of specific Rhokinase inhibitors and their clinical application. Biochim. Biophys. Acta. 1754 (2005) 245–252.

  29. 29.

    Kim, T.Y., Vigil, D., Der, C.J. and Juliano, R.L. Role of DLC-1, a tumor suppressor protein with RhoGAP activity, in regulation of the cytoskeleton and cell motility. Cancer Metastasis Rev. 28 (2009) 77–83.

  30. 30.

    Jaganathan, B.G., Ruester, B., Dressel, L., Stein, S., Grez, M., Seifried, E. and Henschler, R. Rho inhibition induces migration of mesenchymal stromal cells. Stem Cells 25 (2007) 1966–1974.

  31. 31.

    Salhia, B., Rutten, F., Nakada, M., Beaudry, C., Berens, M., Kwan, A. and Rutka, J.T. Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Rac1. Cancer Res. 65 (2005) 8792–8800.

  32. 32.

    Li, Y., Wu, Y., Wang, Z., Zhang, X.H. and Wu, W.K. Fasudil attenuates lipopolysaccharide-induced acute lung injury in mice through the Rho/Rho kinase pathway. Med. Sci. Monit. 16 (2010) BR112–118.

  33. 33.

    Borensztajn, K., Peppelenbosch, M.P. and Spek, C.A. Coagulation Factor Xa inhibits cancer cell migration via LIMK1-mediated cofilin inactivation. Thromb. Res. 125 (2010) e323–328.

  34. 34.

    Koga, T., Awai, M., Tsutsui, J., Yue, B.Y. and Tanihara, H. Rho-associated protein kinase inhibitor, Y-27632, induces alterations in adhesion, contraction and motility in cultured human trabecular meshwork cells. Exp. Eye Res. 82 (2006) 362–370.

  35. 35.

    Honjo, M., Tanihara, H., Kameda, T., Kawaji, T., Yoshimura, N. and Araie, M. Potential role of Rho-associated protein kinase inhibitor Y-27632 in glaucoma filtration surgery. Invest. Ophthalmol. Vis. Sci. 48 (2007) 5549–5557.

  36. 36.

    Kroening, S., Stix, J., Keller, C., Streiff, C. and Goppelt-Struebe, M. Matrixindependent stimulation of human tubular epithelial cell migration by Rho kinase inhibitors. J. Cell Physiol. 223 (2010) 703–712.

  37. 37.

    Tripathi, B.K. and Zelenka, P.S. Cdk5-dependent regulation of Rho activity, cytoskeletal contraction, and epithelial cell migration via suppression of Src and p190RhoGAP. Mol. Cell. Biol. 29 (2009) 6488–6499.

  38. 38.

    Kanda, K., Sobue, K. and Kakiuchi, S. Phosphorylation of myosin light chain and the actin-activated ATPase activity of adrenal medullary myosin. J. Biochem. 97 (1985) 961–964.

  39. 39.

    Shutova, M.S., Alexandrova, A.Y. and Vasiliev, J.M. Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity. Cell. Motil. Cytoskeleton. 65 (2008) 734–746.

  40. 40.

    Palazzo, A.F., Cook, T.A., Alberts, A.S. and Gundersen, G.G. mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol. 3 (2001) 723–729.

  41. 41.

    Niggli, V., Schmid, M. and Nievergelt, A. Differential roles of Rho-kinase and myosin light chain kinase in regulating shape, adhesion, and migration of HT1080 fibrosarcoma cells. Biochem. Biophys. Res. Commun. 343 (2006) 602–608.

  42. 42.

    Smith, A., Bracke, M., Leitinger, B., Porter, J.C. and Hogg, N. LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCKmediated attachment and ROCK-dependent detachment. J. Cell Sci. 116 (2003) 3123–3133.

  43. 43.

    Webb, D.J., Donais, K., Whitmore, L.A., Thomas, S.M., Turner, C.E., Parsons, J.T. and Horwitz, A.F. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 6 (2004) 154–161.

  44. 44.

    Axmann, R., Bohm, C., Kronke, G., Zwerina, J., Smolen, J. and Schett, G. Inhibition of interleukin-6 receptor directly blocks osteoclast formation in vitro and in vivo. Arthritis Rheum. 60 (2009) 2747–2756.

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Correspondence to Xuejiao Zhang or Naoto Kobayashi.

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Zhang, X., Li, C., Gao, H. et al. Rho kinase inhibitors stimulate the migration of human cultured osteoblastic cells by regulating actomyosin activity. Cell Mol Biol Lett 16, 279–295 (2011).

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Key words

  • Osteoblast
  • Migration
  • ROCK inhibitor
  • Cytoskeleton
  • Stress fiber
  • Focal contact
  • MLC phosphorylation