Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells
Coordinated time-dependent modulation of AMPK/Akt/mTOR signaling and autophagy controls osteogenic differentiation of human mesenchymal stem cells
复制标题
DOI：
10.1016/j.bone.2012.10.024
复制
发表时间：
2013-01-01
期刊：
影响因子：
4.1
通讯作者：
Trajkovic, Vladimir
中科院分区：
文献类型：
--
作者：
Pantovic, Aleksandar;Krstic, Aleksandra;Trajkovic, Vladimir
我们研究了AMPK、Akt、mTOR、自噬及其相互作用在人牙髓间充质干细胞成骨分化中的作用。通过免疫印迹分析AMPK、Akt和mTOR信号通路和自噬的各种成员的活化，同时通过碱性磷酸酶染色和骨钙素、Runt相关转录因子2和骨形态发生蛋白2 mRNA和/或蛋白水平的实时RT-PCR/免疫印迹定量评估成骨分化。间充质干细胞的成骨分化与AMPK及其靶Raptor的早期（第1天）激活相关，与mTOR及其底物p70 S6激酶的抑制一致。自噬的早期诱导通过自噬体结合的LC 3 -11的积累、前自噬因子beclin-1的上调和选择性自噬靶点p62的减少来证明。随后是在分化的第3-7天Akt/mTOR的晚期激活。RNA干扰介导的AMPK、mTOR或自噬必需LC 3 β的沉默，以及AMPK（化合物C）、Akt（10-DEBC盐酸盐）、mTOR（雷帕霉素）和自噬（巴弗洛霉素A1、氯喹和氯化铵）的药理学抑制剂，各自抑制间充质干细胞分化成骨细胞。AMPK敲低阻止早期mTOR抑制和自噬诱导，以及Akt/mTOR信号传导的晚期激活，而Ala抑制抑制mTOR激活而不影响AMPK磷酸化。我们的数据表明AMPK通过早期mTOR抑制介导的自噬和晚期Akt/mTOR信号轴激活来控制人间充质干细胞的成骨分化。(C)2012 Elsevier Inc. All rights reserved.
We investigated the role of AMP-activated protein kinase (AMPK), Akt, mammalian target of rapamycin (mTOR), autophagy and their interplay in osteogenic differentiation of human dental pulp mesenchymal stem cells. The activation of various members of AMPK, Akt and mTOR signaling pathways and autophagy was analyzed by immunoblotting, while osteogenic differentiation was assessed by alkaline phosphatase staining and real-time RT-PCR/immunoblot quantification of osteocalcin, Runt-related transcription factor 2 and bone morphogenetic protein 2 mRNA and/or protein levels. Osteogenic differentiation of mesenchymal stem cells was associated with early (day 1) activation of AMPK and its target Raptor, coinciding with the inhibition of mTOR and its substrate p70S6 kinase. The early induction of autophagy was demonstrated by accumulation of autophagosome-bound LC3-11, upregulation of proautophagic,beclin-1 and a decrease in the selective autophagic target p62. This was followed by the late activation of Akt/mTOR at days 3-7 of differentiation. The RNA interference-mediated silencing of AMPK, mTOR or autophagy-essential LC3 beta, as well as the pharmacological inhibitors of AMPK (compound C), Akt (10-DEBC hydrochloride), mTOR (rapamycin) and autophagy (bafilomycin A1, chloroquine and ammonium chloride), each suppressed mesenchymal stem cell differentiation to osteoblasts. AMPK knockdown prevented early mTOR inhibition and autophagy induction, as well as late activation of Akt/mTOR signaling, while Ala inhibition suppressed mTOR activation without affecting AMPK phosphorylation. Our data indicate that AMPK controls osteogenic differentiation of human mesenchymal stem cells through both early mTOR inhibition-mediated autophagy and late activation of Akt/mTOR signaling axis. (C) 2012 Elsevier Inc. All rights reserved.