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

Published online by Cambridge University Press:  02 September 2003

Hyun-A Lee ,
Hyunjoo Jeong ,
Eun-Young Kim ,
Mi Young Nam ,
Yun-Jung Yoo ,
Jeong-Taeg Seo ,
Dong Min Shin ,
Seung-Ho Ohk  and
Syng-Ill Lee
Hyun-A Lee
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Hyunjoo Jeong
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Eun-Young Kim
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Mi Young Nam
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Yun-Jung Yoo
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Jeong-Taeg Seo
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Dong Min Shin
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Seung-Ho Ohk
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
Syng-Ill Lee
Affiliation:
Department of Oral Biology, Department of Pedodontics, Brain Korea 21 Project of Medical Sciences and Oral Science Research Center, Yonsei University College of Dentistry, Seoul 120-749, Korea, and Department of Oral Microbiology, Dental Science Research Institute, Chonnam National University, Gwangju, 501-190, South Korea
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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 multinucleated 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. Experimental Physiology (2003) 88.5, 569-574.

Type
Full Length Papers
Information
Experimental Physiology , Volume 88 , Issue 5 , September 2003 , pp. 569 - 574
Copyright
© The Physiological Society 2003

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