Dehner Manuel, Hadjihannas Michel, Weiske Jörg, Huber Otmar, Behrens Jürgen
Department of Experimental Medicine II, Nikolaus-Fiebiger-Center for Molecular Medicine, University of Erlangen, Glueckstrasse 6, 91054 Erlangen, Germany.
J Biol Chem. 2008 Jul 11;283(28):19201-10. doi: 10.1074/jbc.M710366200. Epub 2008 May 16.
In human cancers, mutations in components of the Wnt signaling pathway lead to beta-catenin stabilization and result in augmented gene transcription. HCT116 colon cancer cells carry stabilizing mutations in beta-catenin and exhibit an elevated activation of Wnt signaling. To clarify the role of an overactive Wnt signaling, we used DNA microarray analysis to search for genes whose expression is up-regulated after knockdown of the wild type adenomatous polyposis coli (APC) tumor suppressor in HCT116 cells, which further enhances Wnt signaling activation. Serum and glucocorticoid-inducible kinase 1 (SGK1) was among the most up-regulated genes following APC knockdown through small interfering RNA. Up-regulation of SGK1 in response to small interfering RNA against APC was inhibited by concomitant knockdown of beta-catenin. Quantitative real time reverse transcription-PCR, Western blot, and chromatin immunoprecipitation analyses confirmed that SGK1 is a direct beta-catenin target gene. SGK1 negatively regulates the pro-apoptotic transcription factor Forkhead box O3a (FoxO3a) via phosphorylation and exclusion from the nucleus. We show that Wnt signaling activation results in FoxO3a exclusion from the nucleus and inhibits expression of FoxO3a target genes. Importantly, FoxO3a mutants that fail to be phosphorylated and therefore are regulated by SGK1 are not influenced by activation of Wnt signaling. In line, knockdown of SGK1 relieves the effects of Wnt signaling on FoxO3a localization and FoxO3a-dependent transcription. Finally, we show that induction of Wnt signaling inhibits FoxO3a-induced apoptosis. Collectively our results indicate that evasion of apoptosis is another feature employed by an overactive Wnt signaling.
在人类癌症中，Wnt信号通路成分的突变导致β-连环蛋白稳定，并导致基因转录增强。HCT116结肠癌细胞在β-连环蛋白中携带稳定突变，并表现出Wnt信号的激活增强。为了阐明过度活跃的Wnt信号的作用，我们使用DNA微阵列分析来寻找在HCT116细胞中野生型腺瘤性息肉病大肠杆菌（APC）肿瘤抑制因子敲低后表达上调的基因，这进一步增强了Wnt信号激活。血清和糖皮质激素诱导激酶1（SGK1）是通过小干扰RNA敲低APC后上调最明显的基因之一。通过小干扰RNA针对APC的反应中SGK1的上调被β-连环蛋白的伴随敲低所抑制。定量实时逆转录-PCR、蛋白质免疫印迹和染色质免疫沉淀分析证实SGK1是直接的β-连环蛋白靶基因。SGK1通过磷酸化和将其排除在细胞核外负向调节促凋亡转录因子叉头框O3a（FoxO3a）。我们表明Wnt信号激活导致FoxO3a从细胞核中排除，并抑制FoxO3a靶基因的表达。重要的是，未能被磷酸化因此受SGK1调节的FoxO3a突变体不受Wnt信号激活的影响。同样，敲低SGK1可减轻Wnt信号对FoxO3a定位和FoxO3a依赖性转录的影响。最后，我们表明Wnt信号的诱导抑制FoxO3a诱导的细胞凋亡。总的来说，我们的结果表明逃避细胞凋亡是过度活跃的Wnt信号采用的另一个特征。
