Barreyro Fernando J, Kobayashi Shogo, Bronk Steven F, Werneburg Nathan W, Malhi Harmeet, Gores Gregory J
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
Division of Gastroenterology and Hepatology, Mayo Clinic College of Medicine, Rochester, Minnesota 55905.
J Biol Chem. 2007 Sep 14;282(37):27141-27154. doi: 10.1074/jbc.M704391200. Epub 2007 Jul 11.
Hepatocyte lipoapoptosis, a critical feature of nonalcoholic steatohepatitis, can be replicated in vitro by incubating hepatocytes with saturated free fatty acids (FFA). These toxic FFA induce Bim expression, which is requisite for their cytotoxicity. Because the FoxO3a transcription factor has been implicated in Bim expression, our aim was to determine if FFA induce Bim by a FoxO3a-dependent mechanism. In Huh-7 cells, the saturated FFA, palmitic and stearic acid, increased Bim mRNA 16-fold. Treatment of cells with the saturated FFA induced FoxO3a dephosphorylation (activation) and nuclear translocation and stimulated a FoxO luciferase-based reporter assay; direct binding of FoxO3a to the Bim promoter was also confirmed by a chromatin immunoprecipitation assay. A small interfering RNA-targeted knockdown of FoxO3a abrogated FFA-mediated Bim induction and apoptosis. FoxO3a was activated by a phosphatase 2A-dependent mechanism, since okadaic acid- and small interfering RNA-targeted knockdown of this phosphatase blocked FoxO3a dephosphorylation, Bim expression, and apoptosis. Consistent with these data, phosphatase 2A activity was also stimulated 3-fold by saturated FFA. Immunoprecpitation studies revealed an FFA-dependent association between FoxO3a and protein phosphatase 2A. FFA-mediated FoxO3a activation by protein phosphatase 2A was also observed in HepG2 cells and murine hepatocytes. In conclusion, saturated FFA stimulate protein phosphatase 2A activity, which activates FoxO3a, inducing expression of the intracellular death mediator Bim.
肝细胞脂肪凋亡是非酒精性脂肪性肝炎的一个关键特征，可通过将肝细胞与饱和游离脂肪酸（FFA）一起孵育在体外复制。这些有毒的FFA诱导Bim表达，这是它们细胞毒性所必需的。由于FoxO3a转录因子与Bim表达有关，我们的目的是确定FFA是否通过FoxO3a依赖的机制诱导Bim。在Huh-7细胞中，饱和FFA棕榈酸和硬脂酸使Bim mRNA增加了16倍。用饱和FFA处理细胞可诱导FoxO3a去磷酸化（激活）和核转位，并刺激基于FoxO荧光素酶的报告基因检测；染色质免疫沉淀检测也证实了FoxO3a与Bim启动子的直接结合。针对FoxO3a的小干扰RNA敲低消除了FFA介导的Bim诱导和凋亡。FoxO3a通过磷酸酶2A依赖的机制被激活，因为冈田酸和针对该磷酸酶的小干扰RNA敲低阻断了FoxO3a去磷酸化、Bim表达和凋亡。与这些数据一致，饱和FFA也使磷酸酶2A活性提高了3倍。免疫沉淀研究揭示了FoxO3a与蛋白磷酸酶2A之间存在FFA依赖的关联。在HepG2细胞和小鼠肝细胞中也观察到蛋白磷酸酶2A介导的FFA对FoxO3a的激活。总之，饱和FFA刺激蛋白磷酸酶2A活性，该活性激活FoxO3a，诱导细胞内死亡介质Bim的表达。
