Department of Anesthesiology and Pain Medicine, School of Medicine, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759, South Korea.
Department of Premedics, School of Medicine, Chosun University, 309 Pilmundaero, Dong-gu, Gwangju 501-759, South Korea.
Biochem Pharmacol. 2015 Dec 1;98(3):511-21. doi: 10.1016/j.bcp.2015.09.007. Epub 2015 Oct 9.
Accumulating evidence suggests that prolyl-isomerase (Pin1) is an important regulator of apoptosis. In the present study, Pin1 was shown to negatively regulate the stabilization of glycogen synthase kinase (GSK) 3αβ serine phosphorylation by inhibiting ubiquitin (Ub)-proteasome degradation, and to induce autophagy following cadmium (Cd) exposure. Cd-induced autophagy in human hepatoma (HepG2) cells has been demonstrated by green fluorescent protein (GFP)-LC3B plasmid DNA transfection, and LC3-II conversion by autophagy inhibitors. Atg5 silencing inhibited Cd-induced apoptosis, indicating that autophagy is involved in cell death. Exposing HepG2 cells to Cd (≤6 μM) initially increased p-Ser GSK3αβ, but then resulted in a gradual decrease, which correlated with polyubiquitinated protein levels, which is indicative of protein degradation. However, high Cd concentrations lead to p-Ser GSK3αβ and polyubiquitinated protein accumulation, indicating proteasome impairment. Cd-induced p-Ser GSK3αβ was enhanced by proteasome inhibition (MG132) and ubiquitin deficiency (cyclohexamide), but no ubiquitination of p-Ser GSK3αβ or GSK3αβ was detected. Cd exposure resulted in p-Ser GSK3αβ redistribution from the nucleus into the cytosol, and this along with autophagy, was inhibited by leptomycin B. Cd concentrations <6 μM did not alter Pin1; however, Cd≥6 μM decreased Pin1. Pin1 overexpression decreased Cd-induced p-Ser GSK3αβ, autophagy, and polyubiquitinated protein levels, while its inhibition reversed the effects. Silencing and overexpression of GSK3αβ had no effect on Cd-induced Pin1 levels. Immunoprecipitation studies showed that Pin1 and p-Ser GSK3αβ interact. Collectively, Pin1 protects cells by inhibiting p-Ser GSK3αβ, and Pin1 decrease upregulates p-Ser GSK3αβ, through the inhibition of Ub-mediated proteasome degradation, and stimulates cell death via autophagy.
越来越多的证据表明脯氨酰异构酶（Pin1）是细胞凋亡的重要调节因子。本研究表明，Pin1 通过抑制泛素（Ub）-蛋白酶体降解负调控糖原合酶激酶（GSK）3αβ丝氨酸磷酸化的稳定，并在镉（Cd）暴露后诱导自噬。通过绿色荧光蛋白（GFP）-LC3B 质粒 DNA 转染和自噬抑制剂的 LC3-II 转化，证实了 Cd 诱导的人肝癌（HepG2）细胞自噬。Atg5 沉默抑制 Cd 诱导的细胞凋亡，表明自噬参与细胞死亡。将 HepG2 细胞暴露于 Cd（≤6 μM）最初增加了 p-Ser GSK3αβ，但随后逐渐减少，这与多聚泛素化蛋白水平相关，表明蛋白降解。然而，高浓度 Cd 导致 p-Ser GSK3αβ 和多聚泛素化蛋白积累，表明蛋白酶体受损。Cd 诱导的 p-Ser GSK3αβ 被蛋白酶体抑制剂（MG132）和泛素缺乏（环己酰胺）增强，但未检测到 p-Ser GSK3αβ 或 GSK3αβ 的泛素化。Cd 暴露导致 p-Ser GSK3αβ 从核内重新分布到细胞质中，这与自噬一起被莱普霉素 B 抑制。Cd 浓度<6 μM 不会改变 Pin1；然而，Cd≥6 μM 会降低 Pin1。Pin1 过表达降低了 Cd 诱导的 p-Ser GSK3αβ、自噬和多聚泛素化蛋白水平，而其抑制作用则逆转了这些作用。GSK3αβ 的沉默和过表达对 Cd 诱导的 Pin1 水平没有影响。免疫沉淀研究表明 Pin1 和 p-Ser GSK3αβ 相互作用。综上所述，Pin1 通过抑制 p-Ser GSK3αβ 来保护细胞，Pin1 减少通过抑制 Ub 介导的蛋白酶体降解而上调 p-Ser GSK3αβ，并通过自噬刺激细胞死亡。
