| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥600,000 (Direct Cost: ¥600,000)
Fiscal Year 2004: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
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| Research Abstract |
ClC-7 Cl^- channel expressing in osteoclasts is important for bone resorption since disruption of its gene in mice leads to a severe osteopetrotic phenotype. However, the functional roles and expression of Cl^- channels other than ClC-7 in osteoclasts are still obscure. In this study, we identified the molecular types of Cl^- channels expressing mouse osteoclasts and examined their functional role in bone resorption.
1.Whole-cell patch-clamp recordings showed that Cl^- current recorded from osteoclasts was characterized by outward rectification, rapid activation and time-dependent inactivation, anion permeability (I>Cl^-), and block by DIDS. These properties of the Cl^- current resembled those of ClC-3 rather than ClC-7. Transcripts for ClC-3 as well as ClC-7 were detected in osteoclasts by RT-PCR. Immunocytochemical analysis also showed the presence of ClC-3 protein in osteoclasts.
2.Reduction of ClC-3 expression in osteoclasts by specific antisense oligonucleotide and small interfering
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RNA (siRNA) against ClC-3 demonstrated a significant reduction in bone resorption activity in vitro. In an in vitro, bone resorption activity of osteoclasts derived from ClC-3-deficient (ClC-3 KO) mice was reduced compared with those from wild type (WT) mice.
3.The Cl^- currents in osteoclasts from ClC-3 KO and WT mice were similar in terms of current kinetics (rapid activation and time-dependent inactivation), densities, rectification, and anion permeability. Furthermore, intracellular dialysis of osteoclasts with anti-ClC-3 antibody did not cause detectable change in the amplitude of outwardly rectifying Cl^- current. These results exclude ClC-3 as the channel responsible for the outwardly rectifying Cl^- current in mouse osteoclasts.
4.Using a pH-sensitive dye, acridine orange, we visualized intracellular acidic compartments (organelles) in living osteoclasts. After treatment with antisense to ClC-3, the number of osteoclasts possessing acidic organelles was significantly decreased. Similar effects were also seen with siRNA against ClC-3. Furthermore, we detected an elevation in the organelle pH of ClC-3 KO osteoclasts, using LysoSensor DND-160, which preferentially partitions into acidic organelles.
These results suggested that mouse osteoclasts expressed ClC-3 and that ClC-3 as well as ClC-7 contributes to maintaining bone resorption activity in vitro. ClC-3 may act as an intracellular Cl^- channel and contribute to acidification of organelles, which lead to the efficient bone resorption. Less
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