UNLABELLED: 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. INTRODUCTION: 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. MATERIALS AND 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. RESULTS AND 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.
UNLABELLED: 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. INTRODUCTION: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. MATERIALS AND 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. RESULTS AND 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.