title: Differential effects of mechanical strain on osteoclastogenesis and             osteoclast-related gene expression in RAW264.7 cells.
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.
authors.affiliation: Institute of Medical Equipment, Academy of Military Medical Sciences, Tianjin 300161, PR China.
authors.forename: Xiao-Ying
authors.initials: XY
authors.lastname: Xu
authors.forename: Chun
authors.initials: C
authors.lastname: Guo
authors.forename: Yu-Xian
authors.initials: YX
authors.lastname: Yan
authors.forename: Yong
authors.initials: Y
authors.lastname: Guo
authors.forename: Rui-Xin
authors.initials: RX
authors.lastname: Li
authors.forename: Mei
authors.initials: M
authors.lastname: Song
authors.forename: Xi-Zheng
authors.initials: XZ
authors.lastname: Zhang
mesh_terms: D000135:Acid Phosphatase / Q000235:genetics / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D001696:Biomechanical Phenomena; D024402:Carbonic Anhydrase II / Q000235:genetics / Q000378:metabolism; D056657:Cathepsin K / Q000235:genetics / Q000378:metabolism; D002452:Cell Count; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D005786:Gene Expression Regulation; D007120:Immunochemistry; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008262:Macrophage Activation; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D053246:Receptor Activator of Nuclear Factor-kappa B / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013314:Stress, Mechanical; D000071681:Tartrate-Resistant Acid Phosphatase; D013997:Time Factors
chemical_list: D007527:Isoenzymes; D012333:RNA, Messenger; D053246:Receptor Activator of Nuclear Factor-kappa B; C505687:Tnfrsf11a protein, mouse; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D056657:Cathepsin K; C534132:Ctsk protein, mouse; D020780:Matrix Metalloproteinase 9; C501830:Mmp9 protein, mouse; D024402:Carbonic Anhydrase II