Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis

Seicho Makihira, Hiroki Nikawa, Mikihito Kajiya, Toshihisa Kawai, Yuichi Mine, Eduardo Kosaka, Marcelo J.B. Silva, Kei Tobiume, Yoshihiro Terada

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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.

Original languageEnglish
Pages (from-to)409-418
Number of pages10
JournalEuropean Journal of Pharmacology
Volume670
Issue number2-3
DOIs
Publication statusPublished - Nov 30 2011

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Vanadates
Cell Fusion
Osteoclasts
Ouabain
Osteogenesis
Adenosine Triphosphatases
Potassium
Sodium
Ions
Bone Marrow Cells
Bone Resorption
Cell Nucleus
Messenger RNA
Ion Exchange
RNA Interference
Real-Time Polymerase Chain Reaction
Cell Culture Techniques
sodium-translocating ATPase

All Science Journal Classification (ASJC) codes

  • Pharmacology

Cite this

Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis. / Makihira, Seicho; Nikawa, Hiroki; Kajiya, Mikihito; Kawai, Toshihisa; Mine, Yuichi; Kosaka, Eduardo; Silva, Marcelo J.B.; Tobiume, Kei; Terada, Yoshihiro.

In: European Journal of Pharmacology, Vol. 670, No. 2-3, 30.11.2011, p. 409-418.

Research output: Contribution to journalArticle

Makihira, Seicho ; Nikawa, Hiroki ; Kajiya, Mikihito ; Kawai, Toshihisa ; Mine, Yuichi ; Kosaka, Eduardo ; Silva, Marcelo J.B. ; Tobiume, Kei ; Terada, Yoshihiro. / Blocking of sodium and potassium ion-dependent adenosine triphosphatase-α1 with ouabain and vanadate suppresses cell-cell fusion during RANKL-mediated osteoclastogenesis. In: European Journal of Pharmacology. 2011 ; Vol. 670, No. 2-3. pp. 409-418.
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AU - Mine, Yuichi

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AU - Silva, Marcelo J.B.

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