Expression of mouse osteoclast K-Cl Co-transporter-1 and its role during bone resorption.
To assess the role of Cl- transport during osteoclastic bone resorption, we studied the expression and function of K+/Cl- co-transporters (KCCs). KCC1 and chloride channel-7 were found to be expressed in mouse osteoclasts. The KCC inhibitor, R(+)-butylindazone (DIOA), KCC1 antisense oligo-nucleotides, and siRNA suppressed osteoclastic pit formation. DIOA also decreased Cl- extrusion and reduced H+ extrusion activity. These results show that KCC1 provides a Cl- extrusion mechanism accompanying the H+ extrusion during bone resorption.


BACKGROUND
Mice with deficient chloride (Cl-) channels, ClC7, show severe osteopetrosis, resulting from impairment of Cl- extrusion during osteoclastic bone resorption. However, the expression and functional role of Cl- transporters other than ClC7 in mammalian osteoclasts is unknown. The aim of this study was to determine expression of K+/Cl- co-transporters (KCCs) and their functional role for bone resorption in mouse osteoclasts.


METHODS
Mouse osteoclasts were derived from cultured bone marrow cells with macrophage-colony stimulating factor (M-CSF) and RANKL or from co-culture of bone marrow cells and primary osteoblasts. We examined the expression of Cl- transporters using RT-PCR, immunochemical, and Western blot methods. The effects of Cl- transport inhibitors on H+ and Cl- extrusion were assessed by measuring intracellular H+ ([H+]i) and Cl- ([Cl-]i). The effects of inhibitors, antisense oligo-nucleotides, and siRNA for Cl- transporters on bone resorption activities were evaluated using a pit formation assay.


CONCLUSIONS
Mouse osteoclasts express not only ClC7 but also K+/Cl- co-transporter mRNA. The existence of KCC1 in the cell membrane of mouse osteoclasts was confirmed by immunochemical staining and Western blot analysis. KCC inhibitors and Cl- channels blockers increased [Cl-]i and [H+]i in resorbing osteoclasts, suggesting that the suppression of Cl- extrusion through KCC and Cl- channels leads to reduced H+ extrusion activity. The combination of both inhibitors greatly suppressed these extrusion activities. KCC inhibitors and Cl- channel blockers also decreased osteoclastic bone resorption in our pit area essay. Furthermore, KCC1 antisense oligo-nucleotides and siRNA suppressed osteoclastic pit formation as well as treatment of ClC7 inhibitors. These results indicate that K+/Cl- co-transporter-1 expressed in mouse osteoclasts acts as a Cl- extruder and plays an important role for H+ extrusion during bone resorption.
Kajiya
Hiroshi
H
Department of Physiological Science and Molecular Biology, Fukuoka Dental College, Tamura, Japan. kajiya@college.fdcnet.ac.jp
Okamoto
Fujio
F
Li
Jing-Ping
JP
Nakao
Akihiro
A
Okabe
Koji
K
D000818:Animals; D001862:Bone Resorption / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000172:deficiency; D018920:Coculture Techniques; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D051379:Mice; D020319:Oligodeoxyribonucleotides, Antisense / Q000235:genetics / Q000494:pharmacology; D010006:Osteoblasts / Q000166:cytology / Q000378:metabolism; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D010022:Osteopetrosis / Q000235:genetics / Q000378:metabolism; D027981:Symporters / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000172:deficiency; D000096922:K Cl- Cotransporters
D018118:Chloride Channels; D020319:Oligodeoxyribonucleotides, Antisense; D027981:Symporters; D000096922:K Cl- Cotransporters; C490803:Clcn7 protein, mouse