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

Clcn7转运体对破骨细胞骨吸收至关重要，其缺乏或突变会抑制骨吸收并导致严重的骨质疏松。法尼酯二磷酸合成酶（Fdps）在甲羟戊酸代谢中催化一种重要的细胞中间体的形成。此外，成熟破骨细胞中的Fdps被含氮双膦酸盐抑制。在这里，我们描述了该酶在破骨细胞Clcn7 cl转运蛋白活性中的新作用。利用酵母双杂交和共免疫沉淀方法，我们在小鼠破骨细胞和过表达HEK细胞中证实了Fdps和Clc7的结合。双膦酸盐可直接抑制骨吸收和促进成熟破骨细胞凋亡。此外，含氮双膦酸盐的主要药理机制是抑制Fdps。因此，我们研究了Fdps抑制剂唑来膦酸对破骨细胞Clcn7挤压活性的影响。细胞外酸化诱导与Clcn7相关的cl -电流，Clcn7在破骨细胞分化过程中被上调。唑来膦酸呈剂量依赖性地抑制酸激活的cl -电流。相反，非氮双膦酸酯对酸激活的cl电流没有影响。四环素诱导的Fdps沉默显著降低了cl电流。此外，cl -电流还抑制了香叶基转移酶抑制剂。与此相反，唑来膦酸的抑制作用被加入甲羟丁酸的衍生物香叶基香叶酸所恢复。虽然细胞外酸化降低了[Cl-] i，但唑来膦酸明显抑制了[Cl-] i的降低。结果表明，含氮双膦酸盐不仅抑制了破骨细胞骨架结构的吸收，还通过抑制成熟破骨细胞的Fdps来抑制酸挤压转运蛋白。

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.