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

BACKGROUND Osteoclast precursor cells are constitutively differentiated into mature osteoclasts on bone tissues. We previously reported that the continuous stimulation of RAW264.7 precursor cells with compressive force induces the formation of multinucleated giant cells via receptor activator of nuclear factor kappaB (RANK)-RANK ligand (RANKL) signaling. Here, we examined the bone resorptive function of multinucleated osteoclasts induced by continuous compressive force. MATERIAL AND METHODS Cells were continuously stimulated with 0.3, 0.6, and 1.1 g/cm² compressive force created by increasing the amount of the culture solution in the presence of RANKL. Actin ring organization was evaluated by fluorescence microscopy. mRNA expression of genes encoding osteoclastic bone resorption-related enzymes was examined by quantitative real-time reverse transcription-polymerase chain reaction. Mineral resorption was evaluated using calcium phosphate-coated plates. RESULTS Multinucleated osteoclast-like cells with actin rings were observed for all three magnitudes of compressive force, and the area of actin rings increased as a function of the applied force. Carbonic anhydrase II expression as well as calcium elution from the calcium phosphate plate was markedly higher after stimulation with 0.6 and 1.1 g/cm² force than 0.3 g/cm². Matrix metalloproteinase-9 expression decreased and cathepsin K expression increased slightly by the continuous application of compressive force. CONCLUSIONS Our study demonstrated that multinucleated osteoclast-like cells induced by the stimulation of RAW264.7 cells with continuous compressive force exhibit high dissolution of the inorganic phase of bone by upregulating carbonic anhydrase II expression and actin ring formation. These findings improve our understanding of the role of mechanical load in bone remodeling.

Continuous application of compressive force induces fusion of osteoclast-like RAW264.7 cells via upregulation of RANK and downregulation of LGR4.

AIMS: During orthodontic treatment, facilitating osteoclastic bone resorption in the alveolar bone exposed to the compressive force (CF) is an important factor for tooth movement. The present study investigated the effect of CF stimulation on the differentiation of RAW264.7 cells from precursors to mature osteoclasts. MAIN METHODS: The cells were continuously stimulated with 0.3, 0.6, or 1.1 g/cm2 CF-which was generated by increasing the volume of culture medium in the wells of a 96-well plate-in the presence or absence of receptor activator of nuclear factor κB (RANK) ligand (RANKL) for 4 days. KEY FINDINGS: In the presence of RANKL, the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells and the mRNA levels of dendritic cell-specific transmembrane protein (DC-STAMP) and osteoclast-stimulatory transmembrane protein (OC-STAMP) were increased by application of 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF. The mRNA level of RANK was upregulated whereas that of leucine-rich repeat-containing G-protein-coupled receptor (LGR)4-another RANKL receptor was downregulated by 0.6 and 1.1 g/cm2 CF as compared to 0.3 g/cm2 CF in the absence of RANKL. The proportion of cells with nuclear translocation of the nuclear translocation of nuclear factor of activated T cells (NFAT)c1 was increased by 0.6 and 1.1 g/cm2 CF in the presence of RANKL. SIGNIFICANCE: Continuous application of CF induced the differentiation of RAW264.7 cells into TRAP-positive multinuclear cells by enhancing the expression of DC- and OC-STAMP and the nuclear translocation of NFATc1. This may result from the CF-induced increase in RANK and decrease in LGR4 expression.

Plasma HDL reduces nonesterified fatty acid hydroperoxides originating from oxidized LDL: a mechanism for its antioxidant ability.

The antioxidant property of plasma high-density lipoprotein (HDL) is thought to be involved in potential anti-atherogenic effects but the exact mechanism is not known. We aimed to reveal the contribution of HDL on the elimination of lipid hydroperoxides (LOOH) derived from oxidized low-density lipoprotein (LDL). Oxidized LDL prepared by copper ion-induced oxidation contained nonesterified fatty acid hydroperoxides (FFA-OOH) and lysophosphatidylcholine (lysoPtdCho), in addition to cholesteryl ester hydroperoxides (CE-OOH) and phosphatidylcholine hydroperoxides (PtdCho-OOH). A platelet-activating factor-acetylhydrolase (PAF-AH) inhibitor suppressed formation of FFA-OOH and lysoPtdCho in oxidized LDL. Among LOOH species, FFA-OOH was preferentially reduced by incubating oxidized LDL with HDL. HDL exhibited selective FFA-OOH reducing ability if it was mixed with a liposomal solution containing FFA-OOH, CE-OOH and PtdCho-OOH. Two-electron reduction of the hydroperoxy group to the hydroxy group was confirmed by the formation of 13-hydroxyoctadecadienoic acid from 13-hydroperoxyoctadecadienoic acid in HPLC analyses. This reducing effect was also found in apolipoprotein A-1 (apoA-1). FFA-OOH released from PtdCho-OOH due to PAF-AH activity in oxidized LDL undergo two-electron reduction by the reducing ability of apoA1 in HDL. This preferential reduction of FFA-OOH may participate in the mechanism of the antioxidant property of HDL.