Regulatory mechanisms of ion transporters expressed in ruffled-border of osteoclasts

我们研究了在吸收破骨细胞的皱褶边缘中激活了何种离子转运。首先，采用全细胞膜片钳技术，比较了在磷酸钙覆盖的盖玻片上培养的有吸收陷窝的大鼠破骨细胞和无吸收破骨细胞的离子电流。然后，外向整流氯电流已被显着激活的破骨细胞的吸收。接下来，我们用RT-PCR方法研究了Cl-转运体在小鼠骨吸收破骨细胞中的表达，以阐明在骨吸收过程中激活的Cl-转运体ClC 3和ClC 7（外向整流Cl-通道）和KCC 1（K^+/Cl^-协同转运体）在破骨细胞中的显著表达。免疫组化染色和Western blot分析证实小鼠破骨细胞膜上存在ClC 7和KCC 1。另一方面，ClC 3主要定位于细胞内细胞器。KCC抑制剂和Cr通道阻断剂增加了再吸收破骨细胞中的[Cl^-] i和[H^+] i，表明通过KCC和Cl通道抑制Cl-排出导致H^+排出活性降低。这两种抑制剂的组合大大抑制了这些挤出活动。KCC抑制剂和Cl-通道阻滞剂也降低了我们的窝区试验中的骨吸收。此外，KCC 1反义寡核苷酸和siRNA抑制骨陷窝形成以及ClC 7抑制剂的治疗。这些结果表明，小鼠破骨细胞表达的ClC 7通道和K^+/Cl^-协同转运蛋白-1在骨吸收过程中起Cl-挤出作用，并在H^+挤出中起重要作用。

We studied what kind of ion transport had been activated in ruffled-border of resorbing osteoclasts. First, the ionic currents of resorbing rat osteoclast with resorption lacuna formed on calcium phosphate courted cover glass were compared with that of non-resorbing osteoclast cultured on the glass cover glass by using whole-cell patch clamp technique. Then, an outward rectifying Cl-current had been remarkably activated in the resorbing osteoclasts. Next, we investigated the Cl-transporter had been expressed in mouse resorbing osteoclasts using RT-PCR method to clarify the Cl-transport activated during bone resorption ClC3 and ClC7 (outward rectifying Cl-channel) and KCC1 (K^+/Cl^-cotransporter) were significantly expressed in osteoclasts. The existences of ClC7 and KCC1 in the cell membrane of mouse osteoclasts were confirmed by immunochemical staining and Western blot analysis. On the other hand, ClC3 was mainly localized in intracellular organelle. KCC inhibitors and Cr-channels blockers increased [Cl^-]_i and [H^+]_i in resorbing osteoclasts, suggesting that the suppression of Cl-extrusion via 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 ClC7 channel and K^+/Cl^- cotransporter-1 expressed in mouse osteoclasts acts as Cl-extruder and plays an important role for H^+ extrusion during bone resorption.