Snx10 and PIKfyve are required for lysosome formation in osteoclasts
Snx10 and PIKfyve are required for lysosome formation in osteoclasts
复制标题
DOI：
10.1002/jcb.29534
复制
发表时间：
2020-04
影响因子：
4
通讯作者：
F. Sultana;L. Morse;G. Picotto;Weimin Liu;P. Jha;P. Odgren;R. Battaglino
中科院分区：
文献类型：
--
作者：
F. Sultana;L. Morse;G. Picotto;Weimin Liu;P. Jha;P. Odgren;R. Battaglino
破骨细胞中的骨吸收和细胞器稳态需要专门的细胞内运输。分选 nexin 10 (Snx10) 是分选 nexin 蛋白家族的成员，通过与位于早期内体的磷酸肌醇 (3) 磷酸 (PI3P) 结合，在内体途径中的货物分选中发挥着至关重要的作用。我们和其他人之前已经证明，编码分选 Snx10 的基因是破骨细胞形态发生和功能所必需的，因为缺乏功能性 Snx10 的人类和小鼠的破骨细胞会出现功能障碍。为了更好地了解 Snx10 调节囊泡运输的作用和机制，本工作的目的是研究另一种 PI3P 结合蛋白 PIKfyve，它将 PI3P 磷酸化为 PI(3,5)P2。已知 PI(3,5)P2 是内体/溶酶体成熟所必需的，并且 PIKfyve 的抑制会导致内体增大。 Snx10 的过度表达还会诱导早期内涵体的积累，表明 Snx10 和 PIKfyve 都是正常内涵体/溶酶体转变所必需的。 Apilimod 是一种小分子，对 PIKfyve 具有特异的纳摩尔级抑制活性，但仅在关键破骨细胞因子 CLCN7、OSTM1 和 Snx10 存在的情况下。这一观察结果表明阿匹莫德的抑制作用是由内体/溶酶体破坏介导的。在这里，我们表明 Snx10 和 PIKfyve 共定位于破骨细胞中的早期内体，并在囊泡组分中共免疫沉淀。用 10 nM 阿匹莫德处理或细胞中 PIKfyve 基因缺失会导致早期内涵体的积累，并抑制破骨细胞的分化、溶酶体形成和分化的破骨细胞分泌 TRAP。 Snx10 和 PIKfyve 还共定位于胃酶原细胞，这是另一种受 Snx10 突变影响的细胞类型。 Apilimod 特异性抑制 PIKfyve 需要 Snx10 表达，因为它不会抑制 Snx10 缺陷破骨细胞中的溶酶体生物发生。这些发现表明，Snx10 和 PIKfyve 通过 PI(3,5)P2 的合成参与内体/溶酶体稳态的调节，并可能指出一种预防骨丢失的新策略。
Bone resorption and organelle homeostasis in osteoclasts require specialized intracellular trafficking. Sorting nexin 10 (Snx10) is a member of the sorting nexin family of proteins that plays crucial roles in cargo sorting in the endosomal pathway by its binding to phosphoinositide(3)phosphate (PI3P) localized in early endosomes. We and others have shown previously that the gene encoding sorting Snx10 is required for osteoclast morphogenesis and function, as osteoclasts from humans and mice lacking functional Snx10 are dysfunctional. To better understand the role and mechanisms by which Snx10 regulates vesicular transport, the aim of the present work was to study PIKfyve, another PI3P‐binding protein, which phosphorylates PI3P to PI(3,5)P2. PI(3,5)P2 is known to be required for endosome/lysosome maturation, and the inhibition of PIKfyve causes endosome enlargement. Overexpression of Snx10 also induces accumulation of early endosomes suggesting that both Snx10 and PIKfyve are required for normal endosome/lysosome transition. Apilimod is a small molecule with specific, nanomolar inhibitory activity on PIKfyve but only in the presence of key osteoclast factors CLCN7, OSTM1, and Snx10. This observation suggests that apilimod's inhibitory effects are mediated by endosome/lysosome disruption. Here we show that both Snx10 and PIKfyve colocalize to early endosomes in osteoclasts and coimmunoprecipitate in vesicle fractions. Treatment with 10 nM apilimod or genetic deletion of PIKfyve in cells resulted in the accumulation of early endosomes, and in the inhibition of osteoclast differentiation, lysosome formation, and secretion of TRAP from differentiated osteoclasts. Snx10 and PIKfyve also colocalized in gastric zymogenic cells, another cell type impacted by Snx10 mutations. Apilimod‐specific inhibition of PIKfyve required Snx10 expression, as it did not inhibit lysosome biogenesis in Snx10‐deficient osteoclasts. These findings suggest that Snx10 and PIKfyve are involved in the regulation of endosome/lysosome homeostasis via the synthesis of PI(3,5)P2 and may point to a new strategy to prevent bone loss.