Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan.
Department of Orthopaedic Surgery, Kenshinkai Kobe Hokuto Hospital, 37-3 Yamada-cho Shimotanigami Aza Umekidani, Kita-ku, Kobe 651-1243, Japan.
Osteoarthritis Cartilage. 2017 Dec;25(12):2134-2146. doi: 10.1016/j.joca.2017.08.019. Epub 2017 Sep 6.
OBJECTIVE: The mammalian target of rapamycin (mTOR) is a serine/threonine kinase that integrates nutrients to execute cell growth and protein synthesis. We hypothesized that mTOR is essential for the intervertebral disc, the largest avascular, low-nutrient organ. Our objective was to elucidate roles of mTOR signaling in human disc cells. DESIGN: The mTOR exists in two complexes: mTORC1 containing the regulatory-associated protein of mTOR (RAPTOR) and mTORC2 containing the rapamycin-insensitive companion of mTOR (RICTOR). To analyze their functions in human disc nucleus pulposus cells, RNA interference (RNAi) of mTOR targeting mTORC1 and mTORC2, RAPTOR targeting mTORC1, or RICTOR targeting mTORC2 or rapamycin, a pharmacological mTORC1 inhibitor, was applied. First, mTOR signaling including Akt, p70/ribosomal S6 kinase (p70/S6K), and autophagy were assessed. Then, apoptosis, senescence, and matrix metabolism were evaluated under pro-inflammatory interleukin-1 beta (IL-1β) stimulation. RESULTS: Western blotting showed significant decreases in specific proteins by each RNAi (all P < 0.0001). In mTOR signaling, RNAi of mTOR and RICTOR decreased p70/S6K and Akt phosphorylation, whereas RAPTOR RNAi decreased p70/S6K but increased Akt phosphorylation. All RNAi treatments increased light chain 3 (LC3)-II and decreased p62/sequestosome 1 (p62/SQSTM1), indicating enhanced autophagy. In apoptosis, IL-1β-induced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells and poly (ADP-ribose) polymerase (PARP) and caspase-9 cleavage decreased by RAPTOR RNAi. In senescence, IL-1β-induced senescence-associated beta-galactosidase (SA-β-gal)-positive cells and p16/INK4A expression also decreased by RAPTOR RNAi. In matrix metabolism, RAPTOR RNAi reduced IL-1β-induced catabolic matrix metalloproteinase (MMP) release and activation and up-regulated anabolic gene expression. These findings were all consistent with rapamycin administration. Additional disc-tissue analysis detected expression and phosphorylation of mTOR-signaling molecules in varying ages. CONCLUSION: Selective interference of mTORC1/RAPTOR protects against inflammation-induced apoptosis, senescence, and matrix catabolism possibly through Akt and autophagy induction in human disc cells.
目的：哺乳动物雷帕霉素靶蛋白（mTOR）是一种丝氨酸/苏氨酸激酶，可整合营养物质以执行细胞生长和蛋白质合成。我们假设 mTOR 对于椎间盘（最大的无血管、低营养器官）至关重要。我们的目的是阐明 mTOR 信号在人椎间盘细胞中的作用。
设计：mTOR 存在于两个复合物中：含有 mTOR 调节相关蛋白（RAPTOR）的 mTORC1 和含有雷帕霉素不敏感伴侣的 mTORC2（RICTOR）。为了分析它们在人椎间盘核髓核细胞中的功能，应用了针对 mTORC1 和 mTORC2 的 mTOR 靶向 RNA 干扰（RNAi）、针对 mTORC1 的 RAPTOR 靶向 RNAi 或针对 mTORC2 的 RICTOR 靶向 RNAi 或雷帕霉素（一种药理学 mTORC1 抑制剂）。首先，评估包括 Akt、p70/核糖体 S6 激酶（p70/S6K）和自噬在内的 mTOR 信号。然后，在促炎白细胞介素 1β（IL-1β）刺激下评估细胞凋亡、衰老和基质代谢。
结果：Western blot 显示每种 RNAi 均显著降低了特定蛋白质的表达（均 P<0.0001）。在 mTOR 信号通路中，mTOR 和 RICTOR 的 RNAi 降低了 p70/S6K 和 Akt 磷酸化，而 RAPTOR RNAi 降低了 p70/S6K 但增加了 Akt 磷酸化。所有 RNAi 处理均增加了微管相关蛋白轻链 3（LC3）-II 并减少了 p62/自噬相关蛋白 1（p62/SQSTM1），表明自噬增强。在细胞凋亡中，RAPTOR RNAi 减少了 IL-1β 诱导的末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）阳性细胞和多聚（ADP-核糖）聚合酶（PARP）和半胱天冬酶-9 切割。在衰老中，RAPTOR RNAi 还减少了 IL-1β 诱导的衰老相关β-半乳糖苷酶（SA-β-gal）阳性细胞和 p16/INK4A 表达。在基质代谢中，RAPTOR RNAi 减少了 IL-1β 诱导的分解代谢基质金属蛋白酶（MMP）释放和激活，并上调了合成代谢基因的表达。这些发现均与雷帕霉素给药一致。对椎间盘组织的进一步分析检测到不同年龄的 mTOR 信号分子的表达和磷酸化。
结论：选择性干扰 mTORC1/RAPTOR 通过诱导 Akt 和自噬来保护人椎间盘细胞免受炎症诱导的细胞凋亡、衰老和基质分解代谢。
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