Bumetanide, the specific inhibitor of Na+-K+-2Cl- cotransport, inhibits 1alpha,25-dihydroxyvitamin D3-induced osteoclastogenesis in a mouse co-culture system.
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)-kappaB ligand (RANKL) and osteoprotegerin (OPG) were analysed by RT-PCR. Exposure of osteoblastic cells to a medium containing 1 micro M bumetanide reduced RANKL mRNA expression induced by 10 nM 1alpha,25-dihydroxyvitamin D3 (1alpha,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 1alpha,25(OH)2D3-induced osteoclastogenesis partly via the phosphorylation of JNK.
Lee
Hyun-A
HA
Department of Oral Biology, Yonsei University College of Dentistry, Seoul 120-749, Korea.
Jeong
Hyunjoo
H
Kim
Eun-Young
EY
Nam
Mi Young
MY
Yoo
Yun-Jung
YJ
Seo
Jeong-Taeg
JT
Shin
Dong Min
DM
Ohk
Seung-Ho
SH
Lee
Syng-Ill
SI
D000818:Animals; D000831:Animals, Newborn; D002034:Bumetanide / Q000494:pharmacology*; D002117:Calcitriol / Q000494:pharmacology*; D002352:Carrier Proteins / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D018920:Coculture Techniques; D004305:Dose-Response Relationship, Drug; D006023:Glycoproteins / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D010010:Osteoclasts / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D018124:Receptors, Tumor Necrosis Factor; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2
D002352:Carrier Proteins; D006023:Glycoproteins; D008562:Membrane Glycoproteins; D053244:Osteoprotegerin; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D018160:Receptors, Cytoplasmic and Nuclear; D018124:Receptors, Tumor Necrosis Factor; C578878:Slc12a2 protein, mouse; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C505687:Tnfrsf11a protein, mouse; C506031:Tnfrsf11b protein, mouse; C505689:Tnfsf11 protein, mouse; D002034:Bumetanide; D002117:Calcitriol