Differential effects of mechanical strain on osteoclastogenesis and osteoclast-related gene expression in RAW264.7 cells

Xu,Xiao-Ying; Guo,Chun; Yan,Yu-Xian; Guo,Yong; Li,Rui-Xin; Song,Mei; Zhang,Xi-Zheng

doi:10.3892/mmr.2012.908

Differential effects of mechanical strain on osteoclastogenesis and osteoclast-related gene expression in RAW264.7 cells

Authors:
- Xiao-Ying Xu
- Chun Guo
- Yu-Xian Yan
- Yong Guo
- Rui-Xin Li
- Mei Song
- Xi-Zheng Zhang
View Affiliations

Affiliations: Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, P.R. China, Department of Orthopaedics, First Affiliated Hospital of Henan University, Kaifeng 475000, Henan, P.R. China
Published online on: May 8, 2012 https://doi.org/10.3892/mmr.2012.908
Pages: 409-415

Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )

Metrics: Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )

Cited By (CrossRef): 0 citations Loading Articles...

Abstract

Mechanical strain plays a critical role in the formation, proliferation and maturation of bone cells. However, little is known about the direct effects of different magnitudes of mechanical strain on osteoclast differentiation. The aim of the present study was to investigate how the fusion and activation of osteoclasts can be regulated by mechanical strain magnitude using the RAW264.7 mouse monocyte/macrophage cell line as an osteoclast precursor. Mechanical strain (substrate stretching) was applied via a 4-point bending system when RAW cells were treated with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB (RANK) ligand (RANKL) for an indicated period of time. The numbers of tartrate-resistant acid phosphatase-positive (TRAP+) and apoptotic cells were counted. The expression of TRAP, matrix metalloproteinase-9 (MMP-9), RANK, cathepsin K and carbonic anhydrase II (CAII) was measured by semi-quantitative RT-PCR, and immunocytochemistry staining for RANK was performed. We found that the number of nuclei per osteoclast derived from RAW cells decreased under low magnitude mechanical strain and increased under high magnitude strain within physiological load with an enhanced fusion of TRAP+ osteoclasts, compared to the control with no mechanical strain. The expression of RANK mRNA was downregulated by low magnitude strain and beyond physiological load, while it was upregulated by high magnitude strain within physiological load, correlating with the increased expression of RANK examined by immunocytochemistry, suggesting the mechanical regulation of RANK expression. There was also an increase in the expression of MMP-9 mRNA in the groups subjected to a mechanical strain of 2,000 and 2,500 µε. No significant differences were detected in the expression of TRAP mRNA, cathepsin K and CAII under mechanical strain compared to the control under no strain (0 µε). These findings indicate that low-magnitude strain suppresses osteoclast fusion and activation, while high-magnitude strain within physiological load promotes osteoclast fusion and activation related to a mechanical magnitude-dependent response of RANK expression. These data, therefore, provide a deeper understanding of how different magnitudes of mechanical strains exert their effects on osteoclastogenesis.

View Figures

View References

1	Frost HM: Bone Remodeling Dynamics. Thomas CC: Springfield, IL: 1963
2	Frost HM: Bone ‘mass’ and the ‘mechanostat’: a proposal. Anat Rec. 219:1–9. 1987.
3	Teitelbaum SL: Osteoclasts: what do they do and how do they do it? Am J Pathol. 170:427–435. 2007.
4	Kodama H, Nose M, Niida S and Yamasaki A: Essential role of macrophage colony-stimulating factor in the osteoclast differentiation supported by stromal cells. J Exp Med. 173:1291–1294. 1991.
5	Yasuda H, Shima N, Nakagawa N, et al: Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro. Endocrinology. 139:1329–1337. 1998.
6	Mochizuki A, Takami M, Kawawa T, et al: Identification and characterization of the precursors committed to osteoclasts induced by TNF-related activation-induced cytokine/receptor activator of NF-kappa B ligand. J Immunol. 177:4360–4368. 2006.
7	Kong YY, Yoshida H, Sarosi I, et al: OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis. Nature. 397:315–323. 1999.
8	Kim N, Odgren PR, Kim DK, Marks SC Jr and Choi Y: Diverse roles of the tumor necrosis factor family member TRANCE in skeletal physiology revealed by TRANCE deficiency and partial rescue by a lymphocyte-expressed TRANCE transgene. Proc Nat Acad Sci USA. 97:10905–10910. 2000.
9	Dougall WC, Glaccum M, Charrier K, et al: RANK is essential for osteoclast and lymph node development. Genes Dev. 13:2412–2424. 1999.
10	Li J, Sarosi I, Yan XQ, et al: RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism. Proc Nat Acad Sci USA. 97:1566–1571. 2000.
11	Chen XY, Zhang XZ, Guo Y, Li RX, Lin JJ and Wei Y: The establishment of a mechanobiology model of bone and functional adaptation in response to mechanical loading. Clin Biomech (Bristol, Avon). 23(Suppl 1): 88–95. 2008.
12	Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT and Martin TJ: Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endoc Rev. 20:345–357. 1999.
13	Suzuki N, Yoshimura Y, Deyama Y, Suzuki K and Kitagawa Y: Mechanical stress directly suppresses osteoclast differentiation in RAW264.7 cells. Int J Mol Med. 21:291–296. 2008.
14	Takahashi N, Udagawa N, Tanaka S and Suda T: Generating murine osteoclasts from bone marrow. Methods Mol Med. 80:129–144. 2003.
15	Tang LL, Wang YL, Pan J and Cai SX: The effect of step-wise increased stretching on rat calvarial osteoblast collagen production. J Biomech. 37:157–161. 2004.
16	Shibata K, Yoshimura Y, Kikuiri T, et al: Effect of the release from mechanical stress on osteoclastogenesis in RAW264.7 cells. Int J Mol Med. 28:73–79. 2011.
17	Kreja L, Liedert A, Hasni S, Claes L and Ignatius A: Mechanical regulation of osteoclastic genes in human osteoblasts. Biochem Biophys Res Commun. 368:582–587. 2008.
18	Rubin J, Murphy T, Nanes MS and Fan X: Mechanical strain inhibits expression of osteoclast differentiation factor by murine stromal cells. Am J Physiol. 278:C1126–1132. 2000.
19	Ichimiya H, Takahashi T, Ariyoshi W, Takano H, Matayoshi T and Nishihara T: Compressive mechanical stress promotes osteoclast formation through RANKL expression on synovial cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 103:334–341. 2007.
20	Rubin J, Fan X, Biskobing DM, Taylor WR and Rubin CT: Osteoclastogenesis is repressed by mechanical strain in an in vitro model. J Orthop Res. 17:639–645. 1999.
21	Burger EH, Klein-Nulend J and Smit TH: Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon – a proposal. J Biomech. 36:1453–1459. 2003.
22	McAllister TN, Du T and Frangos JA: Fluid shear stress stimulates prostaglandin and nitric oxide release in bone marrow-derived preosteoclast-like cells. Biochem Biophys Res Commun. 270:643–648. 2000.
23	Kurata K, Uemura T, Nemoto A, et al: Mechanical strain effect on bone-resorbing activity and messenger RNA expressions of marker enzymes in isolated osteoclast culture. J Bone Miner Res. 16:722–730. 2001.
24	Zhang Q, Liang X, Zhu B, et al: Effects of fluid shear stress on mRNA expression of carbonic anhydrase II in polarized rat osteoclasts. Cell Biol Int. 30:714–720. 2006.
25	Childs LM, Paschalis EP, Xing L, et al: In vivo RANK signaling blockade using the receptor activator of NF-κB:Fc effectively prevents and ameliorates wear debris-induced osteolysis via osteoclast depletion without inhibiting osteogenesis. J Bone Miner Res. 17:192–199. 2002.
26	Feeley BT, Liu NQ, Conduah AH, et al: Mixed metastatic lung cancer lesions in bone are inhibited by noggin overexpression and Rank:Fc administration. J Bone Miner Res. 21:1571–1580. 2006.
27	Kim H, Choi HK, Shin JH, et al: Selective inhibition of RANK blocks osteoclast maturation and function and prevents bone loss in mice. J Clin Invest. 119:813–825. 2009.
28	Blavier L and Delaisse JM: Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones. J Cell Sci. 108:3649–3659. 1995.
29	Sato T, Foged NT and Delaissé JM: The migration of purified osteoclasts through collagen is inhibited by matrix metalloproteinase inhibitors. J Bone Miner Res. 13:59–66. 1998.
30	Ishibashi O, Niwa S, Kadoyama K and Inui T: MMP-9 antisense oligodeoxynucleotide exerts an inhibitory effect on osteoclastic bone resorption by suppressing cell migration. Life Sci. 79:1657–1660. 2006.
31	Hill PA, Murphy G, Docherty AJ, et al: The effects of selective inhibitors of matrix metalloproteinases (MMPs) on bone resorption and the identification of MMPs and TIMP-1 in isolated osteoclasts. J Cell Sci. 107(Pt 11): 3055–3064. 1994.
32	Spessotto P, Rossi FM, Degan M, et al: Hyaluronan-CD44 interaction hampers migration of osteoclast-like cells by down-regulating MMP-9. J Cell Biol. 158:1133–1144. 2002.
33	Alatalo SL, Halleen JM, Hentunen TA, Monkkonen J and Vaananen HK: Rapid screening method for osteoclast differentiation in vitro that measures tartrate-resistant acid phosphatase 5b activity secreted into the culture medium. Clin Chem. 46:1751–1754. 2000.
34	Rissanen JP, Suominen MI, Peng Z and Halleen JM: Secreted tartrate-resistant acid phosphatase 5b is a Marker of osteoclast number in human osteoclast cultures and the rat ovariectomy model. Calcif Tissue Int. 82:108–115. 2008.
35	Fujisaki K, Tanabe N, Suzuki N, et al: Receptor activator of NF-kappaB ligand induces the expression of carbonic anhydrase II, cathepsin K, and matrix metalloproteinase-9 in osteoclast precursor RAW264.7 cells. Life Sci. 80:1311–1318. 2007.