Laboratory of Veterinary Biochemistry and Molecular Biology, College of Veterinary Medicine, Chungbuk National University, Cheongju, Chungbuk, Republic of Korea.
Department of Biomedical Engineering, College of Health Science, Yonsei University, Wonju, Kangwon-do, Republic of Korea.
J Pineal Res. 2010 Apr;48(3):204-211. doi: 10.1111/j.1600-079X.2010.00744.x. Epub 2010 Jan 28.
Cyclosporine A (CsA) is a powerful immunosuppressive drug with side effects including the induction of chronic nephrotoxicity including endoplasmic reticulum (ER) stress in tubular cells. Recently, it was reported that autophagy is induced by ER stress and serves to alleviate the associated deleterious effects. In the current study, CsA treatment (0-100 microm) decreased cell survival of rat pituitary GH3 cells in a dose-dependent manner. At concentrations ranging from 1.0 to 10 microm, CsA induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-I and LC3-II. Cells treated with 2.5 microm CsA exhibited cytoplasmic vacuolation, indicating that CsA induces autophagy in rat pituitary GH3 cells. In the presence of 1.0-10 microm CsA, the expression of catalase decreased while that of the ER stress markers, ER luminal binding protein (BiP) and inositol-requiring enzyme 1 alpha (IRE1alpha), increased as compared those levels in untreated cells. These results suggested that CsA-induced autophagy is dependent on ER stress. To determine whether melatonin would protect cells against CsA-induced autophagy, we treated rat pituitary GH3 cells with melatonin in the presence of CsA. Melatonin treatment (100 and 200 microm) suppressed autophagy induced by 2.5 and 5 microm CsA. Furthermore, co-treatment with 100 microm melatonin inhibited LC3-II expression, and increased catalase and phosphorylated p-ERK levels in the presence of 2.5 and 5 microm CsA. BiP and IRE1alpha expression in melatonin-co-treated cells was superior to that in cells treated with 2.5 and 5 microm CsA alone. Thus, melatonin suppresses CsA-mediated autophagy in rat pituitary GH3 cells.
环孢素 A（CsA）是一种强效的免疫抑制剂，具有副作用，包括诱导肾小管细胞内质网（ER）应激的慢性肾毒性。最近有报道称，自噬是由 ER 应激诱导的，有助于减轻相关的有害影响。在本研究中，CsA 处理（0-100μm）以剂量依赖性方式降低大鼠垂体 GH3 细胞的细胞存活率。在 1.0 至 10μm 的浓度范围内，CsA 诱导微管相关蛋白 1 轻链 3（LC3）-I 和 LC3-II 的表达呈剂量依赖性增加。用 2.5μm CsA 处理的细胞表现出细胞质空泡化，表明 CsA 在大鼠垂体 GH3 细胞中诱导自噬。在存在 1.0-10μm CsA 的情况下，过氧化氢酶的表达降低，而内质网应激标志物内质网腔结合蛋白（BiP）和肌醇需求酶 1α（IRE1α）的表达增加与未处理细胞的水平相比。这些结果表明 CsA 诱导的自噬依赖于 ER 应激。为了确定褪黑素是否可以保护细胞免受 CsA 诱导的自噬，我们在 CsA 存在的情况下用褪黑素处理大鼠垂体 GH3 细胞。褪黑素处理（100 和 200μm）抑制了 2.5 和 5μm CsA 诱导的自噬。此外，在存在 2.5 和 5μm CsA 的情况下，用 100μm 褪黑素共同处理可抑制 LC3-II 的表达，并增加过氧化氢酶和磷酸化 p-ERK 的水平。与单独用 2.5 和 5μm CsA 处理的细胞相比，褪黑素共处理细胞中的 BiP 和 IRE1α 表达更优。因此，褪黑素抑制大鼠垂体 GH3 细胞中 CsA 介导的自噬。
