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

Abstract

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 colonystimulating 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 phosphatasepositive (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.

Continuous Compressive Force Induces Differentiation of Osteoclasts with High Levels of Inorganic Dissolution

“…Hayakawa et al [15] previously revealed that the expression of both cathepsin K and matrix metalloproteinase-9 in RAW264.7 cells pre-incubated with RANKL was increased by stimulation with compressive force (~0.3 g/cm 2 ). Xu et al [34] reported that the effects of mechanical strain on the expression of matrix metalloproteinase-9 and cathepsin K differed, with mechanical strains of 2000 and 2500 μɛ inducing matrix metalloproteinase-9 expression, while cathepsin K expression was not affected by mechanical strain of this magnitude. Mechanical stimulation from physical activity, exercise, and adaptive stress contributes to bone tissue homeostasis [4,5].…”

Plasma Membrane Receptors Involved in the Binding and Response of Osteoclasts to Noncellular Components of the Bone

“…Osteoblasts directly respond to mechanical stimulation by changes in numerous signaling networks [ 142 ], although they are less responsive than osteocytes. Osteoclasts have not been studied as extensively, but in general, it has been shown that mechanical stimulation reduces osteoclast formation and bone resorption when other factors are constant, consistent with the theme that mechanical stimulation is bone anabolic [ 143 , 144 , 145 ]. With the recent studies that suggest that osteoclasts are full partners, with osteoblasts and osteocytes, in signaling involved in the maintenance of the bone [ 18 ], new studies examining how mechanical signaling affects intercellular signals generated by osteoclasts are warranted.…”

Regulatory roles of miRNAs 16, 133a, and 223 on osteoclastic bone destruction caused by breast cancer metastasis

“…A human-derived breast cancer cell line, MDA-MB-231 (HTB-26), and a mouse macrophage cell line, RAW264.7 (TIB-71), both obtained from the American Type Culture Collection (ATCC), were used in the present study. The RAW 264.7 cells are known as preosteoclast cells, which readily differentiate into osteoclasts upon exposure to receptor activator for nuclear factor-κB ligand (RANKL) ( 23 ). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 µ g/ml streptomycin (all from Sigma-Aldrich; Merck KGaA), and maintained in a humidified atmosphere with 5% CO 2 at 37°C.…”