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

Clcn7转运蛋白在破骨细胞性骨吸收中起关键作用，其缺陷或突变抑制骨吸收，导致严重的骨化症。法尼基二磷酸合成酶(FDPS)催化甲氧丙酮酸代谢中一种重要的细胞中间体的形成。此外，在成熟的破骨细胞中，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中Clcn7Cl-转运体活性中的新作用。利用酵母双杂交和免疫共沉淀法，我们在小鼠破骨细胞和高表达Fdps和Clc7的HEK细胞中证明了Fdps与Clcn7结合。双膦酸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机制是抑制FDP。因此，我们研究了Fdps抑制剂唑来膦酸对破骨细胞中Clcn7排泄活性的影响。细胞外酸化诱导与Clcn7相关的氯电流，该电流根据破骨细胞的分化而上调。唑来膦酸以剂量依赖方式抑制酸激活的氯电流。相反，非氮双膦酸埃替磷酸对酸激活的氯离子电流没有影响。四环素诱导的Fdps沉默可显著降低氯离子电流。此外，氯电流还抑制了香叶基香叶转移酶的抑制作用。相反，加入甲酸的衍生物香叶基香叶酸可挽救唑来膦酸的抑制作用。尽管胞外酸化降低了[Cl-]i，但唑来膦酸明显抑制了[Cl-]i的下降。结果提示，含氮双膦酸盐不仅抑制破骨细胞骨架结构的吸收，而且通过抑制成熟破骨细胞的Fdps抑制酸排出转运体。

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.