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

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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.

Abbreviations

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

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Correspondence to Xuejiao Zhang or Naoto Kobayashi.

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Key words

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