Lin Ziying, Liu Tie, Kamp David W, Wang Yahong, He Huijuan, Zhou Xu, Li Donghong, Yang Lawei, Zhao Bin, Liu Gang
Clinical Research Center, Affiliated Hospital of Guangdong Medical College, Zhanjiang 524001, China.
Department of Hematology, The Second Affiliated Hospital, Medical School of Xi׳an Jiaotong University, Xi׳an 710004, Shanxi, China.
Free Radic Biol Med. 2014 Jul;72:296-307. doi: 10.1016/j.freeradbiomed.2014.04.004. Epub 2014 Apr 13.
Chrysotile asbestos is closely associated with excess mortality from pulmonary diseases such as lung cancer, mesothelioma, and asbestosis. Although multiple mechanisms in which chrysotile asbestos fibers induce pulmonary disease have been identified, the role of autophagy in human lung epithelial cells has not been examined. In this study, we evaluated whether chrysotile asbestos induces autophagy in A549 human lung epithelial cells and then analyzed the possible underlying molecular mechanism. Chrysotile asbestos induced autophagy in A549 cells based on a series of biochemical and microscopic autophagy markers. We observed that asbestos increased expression of A549 cell microtubule-associated protein 1 light chain 3 (LC3-II), an autophagy marker, in conjunction with dephosphorylation of phospho-AKT, phospho-mTOR, and phospho-p70S6K. Notably, AKT1/AKT2 double-knockout murine embryonic fibroblasts (MEFs) had negligible asbestos-induced LC3-II expression, supporting a crucial role for AKT signaling. Chrysotile asbestos also led to the phosphorylation/activation of Jun N-terminal kinase (JNK) and p38 MAPK. Pharmacologic inhibition of JNK, but not p38 MAPK, dramatically inhibited the protein expression of LC3-II. Moreover, JNK2(-/-) MEFs but not JNK1(-/-) MEFs blocked LC3-II levels induced by chrysotile asbestos. In addition, N-acetylcysteine, an antioxidant, attenuated chrysotile asbestos-induced dephosphorylation of P-AKT and completely abolished phosphorylation/activation of JNK. Finally, we demonstrated that chrysotile asbestos-induced apoptosis was not affected by the presence of the autophagy inhibitor 3-methyladenine or autophagy-related gene 5 siRNA, indicating that the chrysotile asbestos-induced autophagy may be adaptive rather than prosurvival. Our findings demonstrate that AKT/mTOR and JNK2 signaling pathways are required for chrysotile asbestos-induced autophagy. These data provide a mechanistic basis for possible future clinical applications targeting these signaling pathways in the management of asbestos-induced lung disease.
温石棉与肺癌、间皮瘤和石棉肺等肺部疾病导致的超额死亡率密切相关。尽管已经确定了温石棉纤维诱发肺部疾病的多种机制，但自噬在人肺上皮细胞中的作用尚未得到研究。在本研究中，我们评估了温石棉是否会在A549人肺上皮细胞中诱导自噬，然后分析了可能的潜在分子机制。基于一系列生化和微观自噬标志物，温石棉在A549细胞中诱导了自噬。我们观察到，石棉增加了自噬标志物A549细胞微管相关蛋白1轻链3（LC3-II）的表达，同时伴有磷酸化AKT、磷酸化mTOR和磷酸化p70S6K的去磷酸化。值得注意的是，AKT1/AKT2双敲除小鼠胚胎成纤维细胞（MEF）中石棉诱导的LC3-II表达可忽略不计，这支持了AKT信号传导的关键作用。温石棉还导致了Jun氨基末端激酶（JNK）和p38丝裂原活化蛋白激酶（MAPK）的磷酸化/激活。对JNK而非p38 MAPK的药理学抑制显著抑制了LC3-II的蛋白表达。此外，JNK2（-/-）MEF而非JNK1（-/-）MEF阻断了温石棉诱导的LC3-II水平。此外，抗氧化剂N-乙酰半胱氨酸减弱了温石棉诱导的P-AKT去磷酸化，并完全消除了JNK的磷酸化/激活。最后，我们证明温石棉诱导的凋亡不受自噬抑制剂3-甲基腺嘌呤或自噬相关基因5小干扰RNA（siRNA）的影响，这表明温石棉诱导的自噬可能是适应性的而非促生存的。我们的研究结果表明，AKT/mTOR和JNK2信号通路是温石棉诱导自噬所必需的。这些数据为未来可能针对这些信号通路治疗石棉诱导的肺部疾病提供了机制基础。
