Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis
Transcriptional regulation of Bim by FoxO3A mediates hepatocyte lipoapoptosis
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DOI：
10.1074/jbc.m704391200
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发表时间：
2007-09-14
影响因子：
4.8
通讯作者：
Gores, Gregory J.
中科院分区：
文献类型：
--
作者：
Barreyro, Fernando J.;Kobayashi, Shogo;Gores, Gregory J.
肝细胞脂肪凋亡是非酒精性脂肪性肝炎的一个关键特征，可以通过将肝细胞与饱和游离脂肪酸 (FFA) 一起孵育来在体外复制。这些有毒的 FFA 诱导 Bim 表达，这是其细胞毒性所必需的。由于 FoxO3a 转录因子与 Bim 表达有关，因此我们的目的是确定 FFA 是否通过 FoxO3a 依赖性机制诱导 Bim。在 Huh-7 细胞中，饱和 FFA、棕榈酸和硬脂酸使 Bim mRNA 增加 16 倍。用饱和 FFA 处理细胞会诱导 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测定； FoxO3a 与 Bim 启动子的直接结合也通过染色质免疫沉淀测定得到证实。 FoxO3a 的小干扰 RNA 靶向敲低消除了 FFA 介导的 Bim 诱导和细胞凋亡。 FoxO3a 被磷酸酶 2A 依赖性机制激活，因为冈田酸和小干扰 RNA 靶向敲低该磷酸酶可阻断 FoxO3a 去磷酸化、Bim 表达和细胞凋亡。与这些数据一致，磷酸酶 2A 活性也被饱和 FFA 刺激了 3 倍。免疫沉淀研究揭示了 FoxO3a 和蛋白磷酸酶 2A 之间存在 FFA 依赖性关联。在 HepG2 细胞和鼠肝细胞中也观察到 FFA 介导的蛋白磷酸酶 2A 激活 FoxO3a。总之，饱和 FFA 刺激蛋白磷酸酶 2A 活性，从而激活 FoxO3a，诱导细胞内死亡介质 Bim 的表达。
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.