Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis
Cytosolic entry of bisphosphonate drugs requires acidification of vesicles after fluid-phase endocytosis
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DOI：
10.1124/mol.105.020776
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发表时间：
2006-05-01
影响因子：
3.6
通讯作者：
Crockett, JC
中科院分区：
文献类型：
--
作者：
Thompson, K;Rogers, MJ;Crockett, JC
双膦酸盐如阿仑膦酸盐和唑来膦酸盐是用于抑制破骨细胞介导的骨吸收的重磅炸弹药物。虽然双膦酸盐影响破骨细胞的分子机制现在很明显，但它们被细胞内化的确切途径尚不清楚。为了阐明这一点，我们合成了一种新的，荧光标记的阿仑膦酸钠类似物（AF-ALN）。AF-ALN被迅速内化到J774巨噬细胞和兔破骨细胞的细胞内囊泡中; AF-ALN或[C-14]唑来膦酸盐的摄取被Ca 2+和Sr 2+的存在刺激，并且可以被EGTA或氯膦酸盐抑制，两者都螯合钙离子。EGTA和氯膦酸盐也阻止了双膦酸盐诱导的Rap 1A异戊二烯化抑制，这种作用可通过加入Ca 2+逆转。在J774细胞和破骨细胞中，囊泡AF-ALN与葡聚糖（但不是麦胚凝集素或转铁蛋白）共定位，并且AF-ALN或[C-14]唑来膦酸盐的摄取被丹磺酰尸胺抑制，表明液相内吞参与了双膦酸盐进入囊泡的初始内化。然后，内体酸化似乎是绝对需要的退出囊泡和进入胞质溶胶的双膦酸盐，因为莫能菌素和巴弗洛霉素A1，内体酸化的抑制剂，不抑制囊泡摄取的AF-ALN或内化的[C-14]唑来膦酸盐，但防止阿仑膦酸盐或唑来膦酸盐对Rap 1A异戊二烯化的抑制作用。两者合计，这些结果表明，双膦酸盐药物的细胞摄取需要液相内吞作用，并通过Ca 2+离子增强，而从内吞囊泡转移到胞质溶胶需要内体酸化。
Bisphosphonates such as alendronate and zoledronate are blockbuster drugs used to inhibit osteoclast-mediated bone resorption. Although the molecular mechanisms by which bisphosphonates affect osteoclasts are now evident, the exact route by which they are internalized by cells is not known. To clarify this, we synthesized a novel, fluorescently labeled analog of alendronate (AF-ALN). AF-ALN was rapidly internalized into intracellular vesicles in J774 macrophages and rabbit osteoclasts; uptake of AF-ALN or [C-14]zoledronate was stimulated by the presence of Ca2+ and Sr2+ and could be inhibited by addition of EGTA or clodronate, both of which chelate calcium ions. Both EGTA and clodronate also prevented the bisphosphonate-induced inhibition of Rap1A prenylation, an effect that was reversed by addition of Ca2+. In J774 cells and osteoclasts, vesicular AF-ALN colocalized with dextran ( but not wheat germ agglutinin or transferrin), and uptake of AF-ALN or [C-14] zoledronate was inhibited by dansylcadaverine, indicating that fluid-phase endocytosis is involved in the initial internalization of bisphosphonate into vesicles. Endosomal acidification then seems to be absolutely required for exit of bisphosphonate from vesicles and entry into the cytosol, because monensin and bafilomycin A1, both inhibitors of endosomal acidification, did not inhibit vesicular uptake of AF-ALN or internalization of [C-14] zoledronate but prevented the inhibitory effect of alendronate or zoledronate on Rap1A prenylation. Taken together, these results demonstrate that cellular uptake of bisphosphonate drugs requires fluid-phase endocytosis and is enhanced by Ca2+ ions, whereas transfer from endocytic vesicles into the cytosol requires endosomal acidification.