Analysis of acid extrusion mechanism through new regulatory protein during bone resorption

Clcn 7转运蛋白对骨细胞骨吸收至关重要，其缺陷或突变抑制骨吸收并导致严重的骨硬化症。法呢基二磷酸合酶（Fdps）催化甲羟戊酸代谢中重要的细胞中间体的形成。此外，Fdps在成熟破骨细胞中被含氮双膦酸盐抑制。在这里，我们描述了一种新的作用，在破骨细胞的Clcn 7 Cl-转运活性的酶。使用酵母双杂交和免疫共沉淀方法，我们证明了在小鼠破骨细胞和过表达Fdps和Clc 7的HEK细胞中Fdps与Clc 7的结合。双磷酸盐类药物具有直接抑制骨吸收和促进成熟破骨细胞凋亡的作用。此外，含氮双膦酸盐的主要药理学机制是抑制Fdps。因此，我们研究了Fdps抑制剂唑来膦酸对破骨细胞中Clcn 7挤出活性的影响。细胞外酸化诱导与Clcn 7相关的Cl-电流，Clcn 7根据破骨细胞分化上调。唑来膦酸以剂量依赖性方式抑制酸激活的Cl-电流。相反，非氮双膦酸盐依替膦酸对酸激活的Cl-电流没有影响。四环素诱导的Fdps沉默显著降低Cl-电流。此外，Cl-电流还抑制了香叶基香叶基转移酶的抑制剂。与此相反，唑来膦酸的抑制作用被拯救的另外香叶基香叶酸甲羟戊酸的衍生物。虽然细胞外酸化降低[Cl-] i，但唑来膦酸显著抑制[Cl-] i的降低。结果表明，含氮双膦酸盐不仅抑制再吸收破骨细胞的细胞骨架结构，但也通过抑制Fdps在成熟的破骨细胞抑制酸挤出转运。

The Clcn7 transporter is crucial for osteoclastic bone resorption and its deficiency or mutations suppress bone resorption and lead to severe osteopetrosis. Farnesyl diphosphate synthase(Fdps) catalyses the formation of an important cellular intermediate in mevalonic acid metabolism. Furthermore, Fdps is inhibited by nitrogen-containing bisphosphonate in mature osteoclasts. Here we describe a novel role for the enzyme in Clcn7 Cl-transporter activity in osteoclasts. Using yeast two-hybrid and co-immunoprecipitation methods, we demonstrate the binding of Fdps to Clcn7 in mouse osteoclasts and Fdps and Clc7 overexpressing HEK cells. Bisphosphonates have been attributed to directly inhibit bone resorption and to promote apoptosis of mature osteoclasts. Furthermore, the main pharmacological mechanism of nitrogen-containing bisphosphonates is to inhibit Fdps. Thus, we examined the effects of an Fdps inhibitor zoledronic acid on the Clcn7 extrusion activity in osteoclasts. Extracellular acidification induced Cl-currents associated with Clcn7, which were upregulated in according to osteoclast differentiation. Zoledronic acid inhibited the acid-activated Cl-current in dose-dependent manner. In contrast a non-nitrogen bisphosphonate etidronic acid has no effects on the acid-activated Cl-current. The tetracycline-induced Fdps silencing significantly decreased the Cl-current. Furthermore, the Cl-current also suppressed the inhibitor of geranylgeranyl transferase. In contrast, zoledronic inhibitory effect was rescued by the addition geranylgeranyl acid a derivative of mebalonic acid. Although extracellular acidification decreased[Cl-] i zoledronic acid significantly suppressed the decrease in[Cl-] i. The results suggest that nitrogen-containing bisphosphonates not only suppress resorbing osteoclast cytoskeleton structure but also inhibit acid extrusion transporter through inhibition of Fdps in mature osteoclasts.