Abstract
Fluid flow stress (FSS) is a major mechanical stress that induces bone remodeling upon orthodontic tooth movement, whereas CCN family protein 2 (CCN2) is a potent regenerator of bone defects. In this study, we initially evaluated the effect of laminar FSS on Ccn2 expression and investigated its mechanism in osteoblastic MC3T3-E1 cells. The Ccn2 expression was drastically induced by uniform FSS in an intensity dependent manner. Of note, the observed effect was inhibited by a Rho kinase inhibitor Y27632. Moreover, the inhibition of actin polymerization blocked the FSS-induced activation of Ccn2, whereas inducing F-actin formation using cytochalasin D and jasplakinolide enhanced Ccn2 expression in the same cells. Finally, F-actin formation was found to induce osteoblastic differentiation. In addition, activation of cyclic AMP-dependent kinase, which inhibits Rho signaling, abolished the effect of FSS. Collectively, these findings indicate the critical role of actin polymerization and Rho signaling in CCN2 induction and bone remodeling provoked by FSS.
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Burger EH, Klein-Nulend J (1999) Mechanotransduction in bone–role of the lacuno-canalicular network. FASEB J 13:S101–S112
Cassimeris L, McNeill H, Zigmond SH (1990) Chemoattractant-stimulated polymorphonuclear leukocytes contain two populations of actin filaments that differ in their spatial distributions and relative stabilities. J Cell Biol 110:1067–1075
Cicha I, Goppelt-Struebe M, Muehlich S, Yilmaz A, Raaz D, Daniel WG, Garlichs CD (2008) Pharmacological inhibition of RhoA signaling prevents connective tissue growth factor induction in endothelial cells exposed to non-uniform shear stress. Atherosclerosis 196:136–145
Coue M, Brenner SL, Spector I, Korn ED (1987) Inhibition of actin polymerization by latrunculin A. FEBS Lett 213:316–318
Crean JK, Furlong F, Mitchell D, McArdle E, Godson C, Martin F (2006) Connective tissue growth factor/CCN2 stimulates actin disassembly through Akt/protein kinase B-mediated phosphorylation and cytoplasmic translocation of p27(Kip-1). FASEB J 20:1712–1714
Frangos JA, Eskin SG, McIntire LV, Ives CL (1985) Flow effects on prostacyclin production by cultured human endothelial cells. Science 227:1477–1479
Fu Q, Wu C, Shen Y, Zheng S, Chen R (2008) Effect of LIMK2 RNAi on reorganization of the actin cytoskeleton in osteoblasts induced by fluid shear stress. J Biomech 41:3225–3228
Hahn A, Heusinger-Ribeiro J, Lanz T, Zenkel S, Goppelt-Struebe M (2000) Induction of connective tissue growth factor by activation of heptahelical receptors. Modulation by Rho proteins and the actin cytoskeleton. J Biol Chem 275:37429–37435
Hanna M, Liu H, Amir J, Sun Y, Morris SW, Siddiqui MA, Lau LF, Chaqour B (2009) Mechanical regulation of the proangiogenic factor CCN1/CYR61 gene requires the combined activities of MRTF-A and CREB-binding protein histone acetyltransferase. J Biol Chem 284:23125–23136
Heusinger-Ribeiro J, Eberlein M, Wahab NA, Goppelt-Struebe M (2001) Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP. J Am Soc Nephrol 12:1853–1861
Jackson WM, Jaasma MJ, Tang RY, Keaveny TM (2008) Mechanical loading by fluid shear is sufficient to alter the cytoskeletal composition of osteoblastic cells. Am J Physiol Cell Physiol 295:C1007–C1015
Kubota S, Takigawa M (2007) Role of CCN2/CTGF/Hcs24 in bone growth. Int Rev Cytol 257:1–41
Kubota S, Takigawa M (2011) The role of CCN2 in cartilage and bone development. J Cell Commun Signal 5:209–217
Li J, Zhao Z, Wang J, Chen G, Yang J, Luo S (2008) The role of extracellular matrix, integrins, and cytoskeleton in mechanotransduction of centrifugal loading. Mol Cell Biochem 309:41–48
Moussad EE, Brigstock DR (2000) Connective tissue growth factor: what’s in a name? Mol Genet Metab 71:276–292
Myers KA, Rattner JB, Shrive NG, Hart DA (2007) Osteoblast-like cells and fluid flow: cytoskeleton-dependent shear sensitivity. Biochem Biophys Res Commun 364:214–219
Nishida T, Nakanishi T, Asano M, Shimo T, Takigawa M (2000) Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J Cell Physiol 184:197–206
Ott C, Iwanciw D, Graness A, Giehl K, Goppelt-Struebe M (2003) Modulation of the expression of connective tissue growth factor by alterations of the cytoskeleton. J Biol Chem 278:44305–44311
Perbal B, Takigawa M (2005) CCN protein -A new family of cell growth and differentiation regulators. Imperial College Press, London, pp 1–311
Safadi FF, Xu J, Smock SL, Kanaan RA, Selim AH, Odgren PR, Marks SC Jr, Owen TA, Popoff SN (2003) Expression of connective tissue growth factor in bone: its role in osteoblast proliferation and differentiation in vitro and bone formation in vivo. J Cell Physiol 196:51–62
Sakai Y, Balam TA, Kuroda S, Tamamura N, Fukunaga T, Takigawa M, Takano-Yamamoto T (2009) CTGF and apoptosis in mouse osteocytes induced by tooth movement. J Dent Res 88:345–350
Sin WC, Tse M, Planque N, Perbal B, Lampe PD, Naus CC (2009) Matricellular protein CCN3 (NOV) regulates actin cytoskeleton reorganization. J Biol Chem 284:29935–29944
Smerdel-Ramoya A, Zanotti S, Deregowski V, Canalis E (2008) Connective tissue growth factor enhances osteoblastogenesis in vitro. J Biol Chem 283:22690–22699
Sudo H, Kodama HA, Amagai Y, Yamamoto S, Kasai S (1983) In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria. J Cell Biol 96:191–198
Thi MM, Iacobas DA, Iacobas S, Spray DC (2007) Fluid shear stress upregulates vascular endothelial growth factor gene expression in osteoblasts. Ann N Y Acad Sci 1117:73–81
Weinbaum S, Cowin SC, Zeng Y (1994) A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. J Biomech 27:339–360
Wong GL (1979) Induction of metabolic changes and down regulation of bovine parathyroid hormone-responsive adenylate cyclase are dissociable in isolated osteoclastic and osteoblastic bone cells. J Biol Chem 254:34–37
Woods A, Pala D, Kennedy L, McLean S, Rockel JS, Wang G, Leask A, Beier F (2009) Rac1 signaling regulates CTGF/CCN2 gene expression via TGFbeta/Smad signaling in chondrocytes. Osteoarthr Cartil 17:406–413
Xu J, Millard M, Ren X, Cox OT, Erdreich-Epstein A (2010) c-Abl mediates endothelial apoptosis induced by inhibition of integrins alphavbeta3 and alphavbeta5 and by disruption of actin. Blood 115:2709–2718
Yamashiro T, Fukunaga T, Kobashi N, Kamioka H, Nakanishi T, Takigawa M, Takano-Yamamoto T (2001) Mechanical stimulation induces CTGF expression in rat osteocytes. J Dent Res 80:461–465
Acknowledgments
We thank Dr. M. Miyamoto for providing technical assistance in designing and completing the real time PCR protocols. This study was supported in part by grants-in-aid (20249081T.T-Y) for scientific research from the Japan Society for the Promotion of Science.
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Honjo, T., Kubota, S., Kamioka, H. et al. Promotion of Ccn2 expression and osteoblastic differentiation by actin polymerization, which is induced by laminar fluid flow stress. J. Cell Commun. Signal. 6, 225–232 (2012). https://doi.org/10.1007/s12079-012-0177-z
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DOI: https://doi.org/10.1007/s12079-012-0177-z