Skip to main content
PMC home page
Cellular & Molecular Biology Letters logoLink to Cellular & Molecular Biology Letters
. 2011 Mar 9;16(2):279–295. doi: 10.2478/s11658-011-0006-z

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

Xuejiao Zhang 1,✉, Cheng Li 1, Huiling Gao 1, Hiroaki Nabeka 1, Tetsuya Shimokawa 1, Hiroyuki Wakisaka 1, Seiji Matsuda 1, Naoto Kobayashi 2,✉
PMCID: PMC6275969  PMID: 21394446

Abstract

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.

Key words: Osteoblast, Migration, ROCK inhibitor, Cytoskeleton, Stress fiber, Focal contact, MLC phosphorylation

Full Text

The Full Text of this article is available as a PDF (3.4 MB).

Abbreviations used

CCD

charge-coupled device

EGF

epidermal growth factor

EDTA

ethylenediaminetetraacetic acid

FAK

focal adhesion kinase

FGF

fibroblast growth factor

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

GTP

guanosine triphopsphate

MLC

myosin light chain

MLCK

myosin light chain kinase

PDGF

platelet-derived growth factor

P-MLC

phospharylation of myosin light chain

ROCK

Rho-associated kinase

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

TBST

tris-buffered saline Tween 20

References

  • 1.Williams D.F. On the mechanisms of biocompatibility. Biomaterials. 2008;29:2941–2953. doi: 10.1016/j.biomaterials.2008.04.023. [DOI] [PubMed] [Google Scholar]
  • 2.Wu J., Liu Z.M., Zhao X.H., Gao Y., Hu J., Gao B. Improved biological performance of microarc-oxidized low-modulus Ti-24Nb-4Zr-7.9Sn alloy. J. Biomed. Mater. Res. B Appl. Biomater. 2009;92B:298–306. doi: 10.1002/jbm.b.31515. [DOI] [PubMed] [Google Scholar]
  • 3.Park J.W., Kim H.K., Kim Y.J., Jang J.H., Song H., Hanawa T. Osteoblast response and osseointegration of a Ti-6Al-4V alloy implant incorporating strontium. Acta Biomater. 2010;7:2843–2851. doi: 10.1016/j.actbio.2010.01.017. [DOI] [PubMed] [Google Scholar]
  • 4.Alves S.F., Wassall T. In vitro evaluation of osteoblastic cell adhesion on machined osseointegrated implants. Braz. Oral Res. 2009;23:131–136. doi: 10.1590/S1806-83242009000200007. [DOI] [PubMed] [Google Scholar]
  • 5.Gurkan U.A., Cheng X., Kishore V., Uquillas J.A., 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. 2010;94:1070–1079. doi: 10.1002/jbm.a.32783. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Nakamura M., Nagai A., Tanaka Y., Sekijima Y., Yamashita K. Polarized hydroxyapatite promotes spread and motility of osteoblastic cells. J. Biomed. Mater. Res. A. 2009;92:783–790. doi: 10.1002/jbm.a.32404. [DOI] [PubMed] [Google Scholar]
  • 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., Horman S. AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells. Biochem. Biophys. Res. Commun. 2010;396:656–661. doi: 10.1016/j.bbrc.2010.04.151. [DOI] [PubMed] [Google Scholar]
  • 8.Martini F.J., Valdeolmillos M. Actomyosin contraction at the cell rear drives nuclear translocation in migrating cortical interneurons. J. Neurosci. 2010;30:8660–8670. doi: 10.1523/JNEUROSCI.1962-10.2010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Schwartz M.A., Horwitz A.R. Integrating adhesion, protrusion, and contraction during cell migration. Cell. 2006;125:1223–1225. doi: 10.1016/j.cell.2006.06.015. [DOI] [PubMed] [Google Scholar]
  • 10.Kaibuchi K., Kuroda S., Amano M. Regulation of the cytoskeleton and cell adhesion by the Rho family GTPases in mammalian cells. Annu. Rev. Biochem. 1999;68:459–486. doi: 10.1146/annurev.biochem.68.1.459. [DOI] [PubMed] [Google Scholar]
  • 11.Hall A. Rho GTPases and the actin cytoskeleton. Science. 1998;279:509–514. doi: 10.1126/science.279.5350.509. [DOI] [PubMed] [Google Scholar]
  • 12.Gallagher P.J., Herring B.P., Stull J.T. Myosin light chain kinases. J. Muscle Res. Cell Motil. 1997;18:1–16. doi: 10.1023/A:1018616814417. [DOI] [PubMed] [Google Scholar]
  • 13.Tokuda H., Takai S., Matsushima-Nishiwaki R., Hanai Y., Adachi S., Minamitani C., Mizutani J., Otsuka T., Kozawa O. Function of Rhokinase in prostaglandin D2-induced interleukin-6 synthesis in osteoblasts. Prostaglandins Leukot. Essent. Fatty Acids. 2008;79:41–46. doi: 10.1016/j.plefa.2008.07.004. [DOI] [PubMed] [Google Scholar]
  • 14.Harmey D., Stenbeck G., Nobes C.D., Lax A.J., Grigoriadis A.E. Regulation of osteoblast differentiation by Pasteurella multocida toxin (PMT): a role for Rho GTPase in bone formation. J. Bone Miner. Res. 2004;19:661–670. doi: 10.1359/JBMR.040105. [DOI] [PubMed] [Google Scholar]
  • 15.Kazmers N.H., Ma S.A., Yoshida T., Stern P.H. R. 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. 2009;45:52–60. doi: 10.1016/j.bone.2009.03.675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Loirand G., Guerin P., Pacaud P. Rho kinases in cardiovascular physiology and pathophysiology. Circ. Res. 2006;98:322–334. doi: 10.1161/01.RES.0000201960.04223.3c. [DOI] [PubMed] [Google Scholar]
  • 17.Zohrabian V.M., Forzani B., Chau Z., Murali R., Jhanwar-Uniyal M. Rho/ROCK and MAPK signaling pathways are involved in glioblastoma cell migration and proliferation. Anticancer Res. 2009;29:119–123. [PubMed] [Google Scholar]
  • 18.Hahmann C., Schroeter T. Rho-kinase inhibitors as therapeutics: from pan inhibition to isoform selectivity. Cell. Mol. Life Sci. 2010;67:171–177. doi: 10.1007/s00018-009-0189-x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Lingor P., Teusch N., Schwarz K., Mueller R., Mack H., Bahr M., 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. 2007;103:181–189. doi: 10.1111/j.1471-4159.2007.04756.x. [DOI] [PubMed] [Google Scholar]
  • 20.Ikebe M., Hartshorne D.J. Phosphorylation of smooth muscle myosin at two distinct sites by myosin light chain kinase. J. Biol. Chem. 1985;260:10027–10031. [PubMed] [Google Scholar]
  • 21.Totsukawa G., Yamakita Y., Yamashiro S., Hartshorne D.J., Sasaki Y., 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. 2000;150:797–806. doi: 10.1083/jcb.150.4.797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 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., Kobayashi N. Rho-ROCK signal pathway regulates microtubule-based process formation of cultured podocytes—inhibition of ROCK promoted process elongation. Nephron Exp. Nephrol. 2004;97:e49–61. doi: 10.1159/000078406. [DOI] [PubMed] [Google Scholar]
  • 23.Li C.Y., Gao S.Y., Terashita T., Shimokawa T., Kawahara H., Matsuda S., Kobayashi N. In vitro assays for adhesion and migration of osteoblastic cells (Saos-2) on titanium surfaces. Cell Tissue Res. 2006;324:369–375. doi: 10.1007/s00441-005-0153-5. [DOI] [PubMed] [Google Scholar]
  • 24.Celotti F., Colciago A., Negri-Cesi P., Pravettoni A., Zaninetti R., 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. 2006;14:195–202. doi: 10.1111/j.1743-6109.2006.00110.x. [DOI] [PubMed] [Google Scholar]
  • 25.Tsiridis E., Upadhyay N., Giannoudis P. Molecular aspects of fracture healing: which are the important molecules? Injury. 2007;38(Suppl1):S11–25. doi: 10.1016/j.injury.2007.02.006. [DOI] [PubMed] [Google Scholar]
  • 26.Klein M.O., Reichert C., Koch D., Horn S., 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. 2007;18:40–45. doi: 10.1111/j.1600-0501.2006.01279.x. [DOI] [PubMed] [Google Scholar]
  • 27.Jacobs M., Hayakawa K., Swenson L., Bellon S., Fleming M., Taslimi P., Doran J. The structure of dimeric ROCK I reveals the mechanism for ligand selectivity. J. Biol. Chem. 2006;281:260–268. doi: 10.1074/jbc.M508847200. [DOI] [PubMed] [Google Scholar]
  • 28.Tamura M., Nakao H., Yoshizaki H., Shiratsuchi M., Shigyo H., Yamada H., Ozawa T., Totsuka J., Hidaka H. Development of specific Rhokinase inhibitors and their clinical application. Biochim. Biophys. Acta. 2005;1754:245–252. doi: 10.1016/j.bbapap.2005.06.015. [DOI] [PubMed] [Google Scholar]
  • 29.Kim T.Y., Vigil D., Der C.J., 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. 2009;28:77–83. doi: 10.1007/s10555-008-9167-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Jaganathan B.G., Ruester B., Dressel L., Stein S., Grez M., Seifried E., Henschler R. Rho inhibition induces migration of mesenchymal stromal cells. Stem Cells. 2007;25:1966–1974. doi: 10.1634/stemcells.2007-0167. [DOI] [PubMed] [Google Scholar]
  • 31.Salhia B., Rutten F., Nakada M., Beaudry C., Berens M., Kwan A., Rutka J.T. Inhibition of Rho-kinase affects astrocytoma morphology, motility, and invasion through activation of Rac1. Cancer Res. 2005;65:8792–8800. doi: 10.1158/0008-5472.CAN-05-0160. [DOI] [PubMed] [Google Scholar]
  • 32.Li Y., Wu Y., Wang Z., Zhang X.H., Wu W.K. Fasudil attenuates lipopolysaccharide-induced acute lung injury in mice through the Rho/Rho kinase pathway. Med. Sci. Monit. 2010;16:BR112–118. [PubMed] [Google Scholar]
  • 33.Borensztajn K., Peppelenbosch M.P., Spek C.A. Coagulation Factor Xa inhibits cancer cell migration via LIMK1-mediated cofilin inactivation. Thromb. Res. 2010;125:e323–328. doi: 10.1016/j.thromres.2010.02.018. [DOI] [PubMed] [Google Scholar]
  • 34.Koga T., Awai M., Tsutsui J., Yue B.Y., 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. 2006;82:362–370. doi: 10.1016/j.exer.2005.07.006. [DOI] [PubMed] [Google Scholar]
  • 35.Honjo M., Tanihara H., Kameda T., Kawaji T., Yoshimura N., Araie M. Potential role of Rho-associated protein kinase inhibitor Y-27632 in glaucoma filtration surgery. Invest. Ophthalmol. Vis. Sci. 2007;48:5549–5557. doi: 10.1167/iovs.07-0878. [DOI] [PubMed] [Google Scholar]
  • 36.Kroening S., Stix J., Keller C., Streiff C., Goppelt-Struebe M. Matrixindependent stimulation of human tubular epithelial cell migration by Rho kinase inhibitors. J. Cell Physiol. 2010;223:703–712. doi: 10.1002/jcp.22079. [DOI] [PubMed] [Google Scholar]
  • 37.Tripathi B.K., Zelenka P.S. Cdk5-dependent regulation of Rho activity, cytoskeletal contraction, and epithelial cell migration via suppression of Src and p190RhoGAP. Mol. Cell. Biol. 2009;29:6488–6499. doi: 10.1128/MCB.01098-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kanda K., Sobue K., Kakiuchi S. Phosphorylation of myosin light chain and the actin-activated ATPase activity of adrenal medullary myosin. J. Biochem. 1985;97:961–964. doi: 10.1093/oxfordjournals.jbchem.a135138. [DOI] [PubMed] [Google Scholar]
  • 39.Shutova M.S., Alexandrova A.Y., Vasiliev J.M. Regulation of polarity in cells devoid of actin bundle system after treatment with inhibitors of myosin II activity. Cell. Motil. Cytoskeleton. 2008;65:734–746. doi: 10.1002/cm.20295. [DOI] [PubMed] [Google Scholar]
  • 40.Palazzo A.F., Cook T.A., Alberts A.S., Gundersen G.G. mDia mediates Rho-regulated formation and orientation of stable microtubules. Nat. Cell Biol. 2001;3:723–729. doi: 10.1038/35087035. [DOI] [PubMed] [Google Scholar]
  • 41.Niggli V., Schmid M., 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. 2006;343:602–608. doi: 10.1016/j.bbrc.2006.03.022. [DOI] [PubMed] [Google Scholar]
  • 42.Smith A., Bracke M., Leitinger B., Porter J.C., Hogg N. LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCKmediated attachment and ROCK-dependent detachment. J. Cell Sci. 2003;116:3123–3133. doi: 10.1242/jcs.00606. [DOI] [PubMed] [Google Scholar]
  • 43.Webb D.J., Donais K., Whitmore L.A., Thomas S.M., Turner C.E., Parsons J.T., Horwitz A.F. FAK-Src signalling through paxillin, ERK and MLCK regulates adhesion disassembly. Nat. Cell Biol. 2004;6:154–161. doi: 10.1038/ncb1094. [DOI] [PubMed] [Google Scholar]
  • 44.Axmann R., Bohm C., Kronke G., Zwerina J., Smolen J., Schett G. Inhibition of interleukin-6 receptor directly blocks osteoclast formation in vitro and in vivo. Arthritis Rheum. 2009;60:2747–2756. doi: 10.1002/art.24781. [DOI] [PubMed] [Google Scholar]

Articles from Cellular & Molecular Biology Letters are provided here courtesy of BMC

RESOURCES