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MOLECULAR IDENTIFICATION AND FUNCTIONAL ANALYSIS OF Cl^- CHANNELS CONTRIBUTING OSTEOCLASTIC BONE RESORPTION.

有助于破骨骨吸收的 Cl^- 通道的分子鉴定和功能分析。

基本信息

批准号：
15591988
负责人：
OKAMOTO Fujio
金额：
$ 2.24万
依托单位：
Fukuoka Dental College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2005
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15591988/
关键词：
BONE RESORPTION OSTEOCLAST Cl^- CHANNEL ANTISENSE RNAi ClC-3 KNOCK OUT MICE ACIDIC ORGANELLE ClC-3ノックアウト CLC-3 siRNA 細胞内酸性化

项目摘要

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 … More 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

在破骨细胞中表达的ClC-7 Cl^-通道对于骨吸收是重要的，因为在小鼠中其基因的破坏导致严重的骨硬化表型。然而，除了ClC-7之外，Cl^-通道在破骨细胞中的功能作用和表达仍然不清楚。本研究鉴定了小鼠破骨细胞表达Cl^-通道的分子类型，并研究了其在骨吸收中的功能作用：1.全细胞膜片钳记录表明，破骨细胞记录的Cl^-电流具有外向整流、快速激活和时间依赖性失活、阴离子渗透性（I>Cl^-）和DIDS阻断等特征。Cl^-电流的这些特性与ClC-3的类似，而与ClC-7的不同。通过RT-PCR检测破骨细胞中ClC-3和ClC-7的转录物。免疫细胞化学分析也显示破骨细胞中存在ClC-3蛋白。2.特异性反义寡核苷酸和小分子干扰抑制破骨细胞中ClC-3的表达 ...更多信息针对ClC-3的RNA（siRNA）在体外证明了骨吸收活性的显著降低。在体外实验中，ClC-3敲除（ClC-3 KO）小鼠破骨细胞的骨吸收活性比野生型（WT）小鼠的低。3. ClC-3敲除小鼠和WT小鼠破骨细胞的Cl^-电流在电流动力学（快速激活和时间依赖性失活）、密度、整流和阴离子渗透性方面相似。此外，用抗ClC-3抗体对破骨细胞进行细胞内透析并没有引起向外整流Cl^-电流幅度的可检测变化。这些结果排除了ClC-3作为负责小鼠破骨细胞中向外整流Cl^-电流的通道。4.使用pH敏感染料吖啶橙子，我们观察了活破骨细胞中的细胞内酸性区室（细胞器）。经ClC-3反义核酸处理后，具有酸性细胞器的破骨细胞数量显著减少。用针对ClC-3的siRNA也观察到类似的效果。此外，我们检测到ClC-3 KO破骨细胞的细胞器pH升高，使用LysoSensor DND-160，其优先分配到酸性细胞器中。这些结果表明，小鼠破骨细胞表达ClC-3，ClC-3和ClC-7有助于维持体外骨吸收活性。ClC-3可能作为细胞内Cl^-通道，促进细胞器酸化，从而导致有效的骨吸收。少