title: Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis.
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 β2 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.
authors.affiliation: Section of Fixed Prosthodontics, Department of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka 812-8582, Japan. makihira@dent.kyushu-u.ac.jp
authors.forename: Seicho
authors.initials: S
authors.lastname: Makihira
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nikawa
authors.forename: Mikihito
authors.initials: M
authors.lastname: Kajiya
authors.forename: Toshihisa
authors.initials: T
authors.lastname: Kawai
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Mine
authors.forename: Eduardo
authors.initials: E
authors.lastname: Kosaka
authors.forename: Marcelo J B
authors.initials: MJ
authors.lastname: Silva
authors.forename: Kei
authors.initials: K
authors.lastname: Tobiume
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Terada
mesh_terms: D000135:Acid Phosphatase / Q000378:metabolism; D000818:Animals; D002454:Cell Differentiation / Q000187:drug effects; D002459:Cell Fusion; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D007527:Isoenzymes / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000235:genetics; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects*; D010042:Ouabain / Q000494:pharmacology*; D021122:Protein Subunits / Q000037:antagonists & inhibitors / Q000172:deficiency / Q000235:genetics; D053245:RANK Ligand / Q000737:chemistry / Q000494:pharmacology*; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors* / Q000172:deficiency / Q000235:genetics; D012995:Solubility; D000071681:Tartrate-Resistant Acid Phosphatase; D014638:Vanadates / Q000494:pharmacology*
chemical_list: C499636:DC-STAMP protein, mouse; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D021122:Protein Subunits; D053245:RANK Ligand; D012333:RNA, Messenger; D014638:Vanadates; D010042:Ouabain; D000135:Acid Phosphatase; C000605511:Acp5 protein, mouse; D000071681:Tartrate-Resistant Acid Phosphatase; D000254:Sodium-Potassium-Exchanging ATPase