ABSTRACT
To examine the possible enrolment of Na(+)/K(+)-ATPase during osteoclast differentiation, Na(+)/K(+)-ATPase inhibitors, including ouabain and vanadate, were used in this study. These inhibitors significantly inhibited cell-cell fusion of RAW264.7 cells and bone marrow cells induced by RANKL. Interestingly, in response to RANKL-stimulation, ouabain and vanadate decreased the number of large TRAP+ osteoclasts in the culture of RAW264.7 cells, as well as bone marrow cells. In contrast, the number of small TRAP+ osteoclasts either increased in RAW264.7 cells or were otherwise less affected in bone marrow cells than large TRAP+ osteoclasts. Large TRAP+ osteoclasts are defined as having ≥ 10 nuclei/cell and having more potency in bone resorption than small multinuclear osteoclasts with <9 nuclei/cell. Na(+)/K(+)-ATPase α1 and ß2 mRNAs were detected in sRANKL-stimulated RAW264.7 cells. Moreover, real-time quantitative PCR showed that ouabain and vanadate suppressed the RANKL-dependent induction of the osteoclast fusion-promotion molecule DC-STAMP at the mRNA level. Finally, and importantly, RNAi-mediated suppression of Na(+)/K(+)-ATPase α1 resulted in a diminished number of large TRAP+ osteoclasts in the sRANKL-stimulated RAW264.7 cells, along with the decreased level of DC-STAMP mRNA expression. These findings strongly suggest that blockage of the Na(+)/K(+)-ATPase α1 subunit by ouabain or vanadate caused the inhibition of RANKL-induced cell-cell fusion, resulting in the generation of large osteoclasts through suppression of DC-STAMP expression. Thus, in addition to its known function of sodium and potassium ion exchange during bone resorption by mature osteoclasts, this study has revealed a novel molecular role of the Na(+)/K(+)-ATPase α1 subunit in osteoclastogenesis.
Subject(s)
Enzyme Inhibitors/pharmacology , Osteoclasts/cytology , Osteoclasts/drug effects , Ouabain/pharmacology , RANK Ligand/pharmacology , Sodium-Potassium-Exchanging ATPase/antagonists & inhibitors , Vanadates/pharmacology , Acid Phosphatase/metabolism , Animals , Cell Differentiation/drug effects , Cell Fusion , Cell Line , Gene Expression Regulation, Enzymologic/drug effects , Isoenzymes/metabolism , Membrane Proteins/genetics , Mice , Nerve Tissue Proteins/genetics , Protein Subunits/antagonists & inhibitors , Protein Subunits/deficiency , Protein Subunits/genetics , RANK Ligand/chemistry , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sodium-Potassium-Exchanging ATPase/deficiency , Sodium-Potassium-Exchanging ATPase/genetics , Solubility , Tartrate-Resistant Acid PhosphataseABSTRACT
The purpose of the present study was to examine the effect of osteoprotegerin (OPG)-Fc fusion protein immobilized on a titanium surface on the initial differentiation of osteoclast precursor RAW264.7 cells. These cells were cultured on titanium specimens over which OPG-Fc was immobilized. The enhancement of tartrate-resistant acid phosphatase (TRAP) and cathepsin K mRNA expression in RAW264.7 cells exposed to receptor activator of NF-kappaB ligand (RANKL) stimulation on OPG-Fc-coated titanium was significantly lower than that in RAW264.7 cells exposed to RANKL on titanium specimens without immobilized OPG-Fc (ANOVA, P < 0.01). Preincubation of OPG-Fc-coated titanium, in a medium supplemented with 10% fetal bovine serum at 37 degrees C for two days before the cells were seeded, had no significant effect on the decrease in mRNA expression (ANOVA, P < 0.01). Taken together, these results indicate that OPG-Fc immobilized on a titanium surface blocks the differentiation of RAW264.7 cells induced by RANKL stimulation.
Subject(s)
Coated Materials, Biocompatible/pharmacology , Macrophages/cytology , Macrophages/metabolism , Osteoclasts/cytology , Osteoclasts/metabolism , Osteoprotegerin/chemistry , Osteoprotegerin/pharmacology , RANK Ligand/metabolism , Titanium/chemistry , Adsorption , Animals , Cell Differentiation/drug effects , Cell Line , Coated Materials, Biocompatible/chemistry , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/pharmacology , Macrophages/drug effects , Materials Testing , Mice , Osteoclasts/drug effects , Surface PropertiesABSTRACT
The present study was a molecular analysis of the initial differentiation of osteoclast precursor RAW264.7 cells on titanium specimens. RAW264.7 cell line was cultured on titanium specimens of which the surfaces were finished by wet grinding with 2000-, 1200-, 600-, or 180-grit waterproof abrasive paper. Total RNA was extracted from cells cultured in the presence or absence of Receptor Activator of NF-kappaB Ligand (RANKL), prior to cDNA synthesis for real-time quantitative reverse transcriptase-polymerase chain reaction analysis. Titanium surfaces initially enhanced the expression of osteoclast differentiation markers including tartrate-resistant acid phosphatase and cathepsin K in RAW264.7 cells cultured with RANKL stimulation, in a roughness-dependent manner. The mRNA expressions of both RANKL receptor, RANK, and its adapter protein TNF receptor-associated factor 6 (TRAF6) increased when RAW264.7 cells were cultured on titanium specimens with roughened surfaces, as compared with that of control specimen with a polished surface. These results, taken together, suggested that titanium surface roughness facilitated osteoclast differentiation through the activation of the RANK-TRAF6 signaling network.