Hu Yingwei, Carraro-Lacroix Luciene R, Wang Andrew, Owen Celeste, Bajenova Elena, Corey Paul N, Brumell John H, Voronov Irina
Faculty of Dentistry, University of Toronto, Toronto, ON, Canada.
Institute of Dental Medicine, Qilu Hospital, Shandong University, Jinan, China.
J Cell Biochem. 2016 Feb;117(2):413-25. doi: 10.1002/jcb.25287.
Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in the regulation of cell growth. It has been shown to play an important role in osteoclast differentiation, particularly at the earlier stages of osteoclastogenesis. mTOR activation and function, as part of mTORC1 complex, is dependent on lysosomal localization and the vacuolar H(+) -ATPase (V-ATPase) activity; however, the precise mechanism is still not well understood. Using primary mouse osteoclasts that are known to have higher lysosomal pH due to R740S mutation in the V-ATPase a3 subunit, we investigated the role of lysosomal pH in mTORC1 signaling. Our results demonstrated that +/R740S cells had increased basal mTOR protein levels and mTORC1 activity compared to +/+ osteoclasts, while mTOR gene expression was decreased. Treatment with lysosomal inhibitors chloroquine and ammonium chloride, compounds known to raise lysosomal pH, significantly increased mTOR protein levels in +/+ cells, confirming the importance of lysosomal pH in mTOR signaling. These results also suggested that mTOR could be degraded in the lysosome. To test this hypothesis, we cultured osteoclasts with chloroquine or proteasomal inhibitor MG132. Both chloroquine and MG132 increased mTOR and p-mTOR protein levels in +/+ osteoclasts, suggesting that mTOR undergoes both lysosomal and proteasomal degradation. Treatment with cycloheximide, an inhibitor of new protein synthesis, confirmed that mTOR is constitutively expressed and degraded. These results show that, in osteoclasts, the lysosome plays a key role not only in mTOR activation but also in its deactivation through protein degradation, representing a novel molecular mechanism of mTOR regulation.
雷帕霉素的哺乳动物靶点（mTOR）是一种参与细胞生长调节的丝氨酸/苏氨酸激酶。研究表明，它在破骨细胞分化中起着重要作用，尤其是在破骨细胞生成的早期阶段。作为mTORC1复合物的一部分，mTOR的激活和功能依赖于溶酶体定位和液泡H(+) -ATP酶（V-ATP酶）活性；然而，确切机制仍未完全清楚。我们使用由于V-ATP酶a3亚基中的R740S突变而已知具有较高溶酶体pH值的原代小鼠破骨细胞，研究了溶酶体pH值在mTORC1信号传导中的作用。我们的结果表明，与+/ +破骨细胞相比，+/R740S细胞的基础mTOR蛋白水平和mTORC1活性增加，而mTOR基因表达降低。用溶酶体抑制剂氯喹和氯化铵（已知可提高溶酶体pH值的化合物）处理，显著增加了+/ +细胞中的mTOR蛋白水平，证实了溶酶体pH值在mTOR信号传导中的重要性。这些结果还表明mTOR可能在溶酶体中被降解。为了验证这一假设，我们用氯喹或蛋白酶体抑制剂MG132培养破骨细胞。氯喹和MG132均增加了+/ +破骨细胞中的mTOR和p-mTOR蛋白水平，表明mTOR经历溶酶体和蛋白酶体降解。用新蛋白质合成抑制剂环己酰亚胺处理证实mTOR是组成性表达和降解的。这些结果表明，在破骨细胞中，溶酶体不仅在mTOR激活中起关键作用，而且在通过蛋白质降解使其失活中起关键作用，代表了一种新的mTOR调节分子机制。
