Bumetanide, the specific inhibitor of Na+-K+-2C1- cotransport, inhibits 1α,25-dihydroxyvitamin D3-induced osteoclastogenesis in a mouse co-culture system

Hyun A. Lee, Hyunjoo Jeong, Eun Young Kim, Mi Young Nam, Yun Jung Yoo, Jeong Taeg Seo, Dong Min Shin, Seung Ho Ohk, Syng Ill Lee

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The Na+-K+-2Cl- cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multi-nucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 μM bumetanide reduced RANKL mRNA expression induced by 10 nM 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2-terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1α,25(OH)2D3-induced osteoclastogenesis partly via the phosphorylation of JNK.

Original languageEnglish
Pages (from-to)569-574
Number of pages6
JournalExperimental Physiology
Volume88
Issue number5
DOIs
Publication statusPublished - 2003 Sep

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Bumetanide
Calcitriol
Coculture Techniques
Osteogenesis
Osteoprotegerin
Messenger RNA
JNK Mitogen-Activated Protein Kinases
Osteoblasts
Member 2 Solute Carrier Family 12
Phosphorylation
RANK Ligand
Macrophage Colony-Stimulating Factor
Tumor Necrosis Factor Receptors
Ion Transport
Osteoclasts
Cytoplasmic and Nuclear Receptors
Cell Size
Growth and Development
Anti-Idiotypic Antibodies
Epithelium

All Science Journal Classification (ASJC) codes

  • Physiology
  • Nutrition and Dietetics
  • Physiology (medical)

Cite this

Lee, Hyun A. ; Jeong, Hyunjoo ; Kim, Eun Young ; Nam, Mi Young ; Yoo, Yun Jung ; Seo, Jeong Taeg ; Shin, Dong Min ; Ohk, Seung Ho ; Lee, Syng Ill. / Bumetanide, the specific inhibitor of Na+-K+-2C1- cotransport, inhibits 1α,25-dihydroxyvitamin D3-induced osteoclastogenesis in a mouse co-culture system. In: Experimental Physiology. 2003 ; Vol. 88, No. 5. pp. 569-574.
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abstract = "The Na+-K+-2Cl- cotransporter (NKCC1) is responsible for ion transport across the secretory and absorptive epithelia, the regulation of cell volume, and possibly the modulation of cell growth and development. It has been reported that a variety of cells, including osteoblasts, contain this cotransporter. In this study, the physiological role of NKCC1 in osteoclastogenesis was exploited in a co-culture system. Bumetanide, a specific inhibitor of NKCC1, reduced the number of tartrate-resistant acid phosphatase (TRAP)-positive multi-nucleated cells. In order to investigate the mechanism by which bumetanide inhibits osteoclastogenesis, the mRNA expressions of the receptor activator of nuclear factor (NF)-κB ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 μM bumetanide reduced RANKL mRNA expression induced by 10 nM 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3, in a dose-dependent manner. In addition, RANKL expression was also analysed with enzyme-linked immunosorbant assay (ELISA) using anti-RANKL antibody. The expression of RANKL was decreased with the increase of bumetanide concentration. In contrast, the expression of OPG mRNA, a novel tumour necrosis factor (TNF) receptor family member was increased in the presence of bumetanide. These results imply that bumetanide inhibits osteoclast differentiation by reducing the RANKL/OPG ratio in osteoblastic cells. However, no significant difference in M-CSF mRNA expression was observed when bumetanide was added. Also, we found that the phosphorylation of c-Jun NH2-terminal kinase (JNK), which regulates the activity of various transcriptional factors, was reduced by bumetanide treatment. Conclusively, these findings suggest that NKCC1 in osteoblasts has a pivotal role in 1α,25(OH)2D3-induced osteoclastogenesis partly via the phosphorylation of JNK.",
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Bumetanide, the specific inhibitor of Na+-K+-2C1- cotransport, inhibits 1α,25-dihydroxyvitamin D3-induced osteoclastogenesis in a mouse co-culture system. / Lee, Hyun A.; Jeong, Hyunjoo; Kim, Eun Young; Nam, Mi Young; Yoo, Yun Jung; Seo, Jeong Taeg; Shin, Dong Min; Ohk, Seung Ho; Lee, Syng Ill.

In: Experimental Physiology, Vol. 88, No. 5, 09.2003, p. 569-574.

Research output: Contribution to journalArticle

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AU - Lee, Hyun A.

AU - Jeong, Hyunjoo

AU - Kim, Eun Young

AU - Nam, Mi Young

AU - Yoo, Yun Jung

AU - Seo, Jeong Taeg

AU - Shin, Dong Min

AU - Ohk, Seung Ho

AU - Lee, Syng Ill

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