背景:滋养层稳态和分化需要适当的内质网(ER)功能。Krüppel样因子-6(KLF6)转录因子调节滋养层迁移,分化,和活性氧(ROS)的产生。由于ROS可能会影响ER稳态,我们评估了KLF6的下调是否改变了与ER稳态相关的未折叠蛋白反应(UPR)和细胞过程.
方法:通过Westernblot和qRT-PCR分析蛋白质和RNA表达,分别,在KLF6沉默的绒毛外滋养层HTR-8/SVneo细胞中。使用膜联蛋白V细胞凋亡检测试剂盒通过流式细胞术检测细胞凋亡。通过报告荧光蛋白的共聚焦显微镜评估蛋白质运输,其从ER的释放是同步的。
结果:KLF6下调降低了BiP的表达,普遍定期审议的主要监管者,蛋白质,mRNA和前mRNA水平。Ire1α蛋白,XBP1拼接,在KLF6沉默的细胞中,DNAJB9mRNA水平也降低。相反,PDI,Ero1α,p-eIF2α/eIF2α比值以及自噬和蛋白酶体依赖性蛋白降解保持不变,而细胞内运输增加。在thapsigargin诱导的应激下,KLF6沉默受损的BiP蛋白和mRNA表达增加,以及Ire1α途径的激活,但它提高了p-eIF2α/eIF2α比值和CHOP蛋白水平。然而,细胞凋亡没有增加。
结论:结果提供了KLF6作为UPR组分调节剂的第一个证据。蛋白质运输和细胞凋亡保护的增加,在KLF6沉默的细胞中观察到,与其在绒毛外滋养层迁移和分化中的作用一致。
BACKGROUND: Trophoblast homeostasis and differentiation require a proper endoplasmic reticulum (ER) function. The Krüppel-like factor-6 (KLF6) transcription factor modulates trophoblast migration, differentiation, and reactive oxygen species (ROS) production. Since ROS may impact on ER homeostasis, we assessed whether downregulation of KLF6 altered the unfolded protein response (UPR) and cellular process associated with ER homeostasis.
METHODS: Protein and RNA expression were analyzed by Western blot and qRT-PCR, respectively, in extravillous trophoblast HTR-8/SVneo cells silenced for KLF6. Apoptosis was detected by flow cell cytometry using Annexin V Apoptosis Detection Kit. Protein trafficking was assessed by confocal microscopy of a reporter fluorescent protein whose release from the ER was synchronized.
RESULTS: KLF6 downregulation reduced the expression of BiP, the master regulator of the UPR, at protein, mRNA, and pre-mRNA levels. Ire1α protein, XBP1 splicing, and DNAJB9 mRNA levels were also reduced in KLF6-silenced cells. Instead, PDI, Ero1α, and the p-eIF2α/eIF2α ratio as well as autophagy and proteasome dependent protein degradation remained unchanged while intracellular trafficking was increased. Under thapsigargin-induced stress, KLF6 silencing impaired BiP protein and mRNA expression increase, as well as the activation of the Ire1α pathway, but it raised the p-eIF2α/eIF2α ratio and CHOP protein levels. Nevertheless, apoptosis was not increased.
CONCLUSIONS: Results provide the first evidence of KLF6 as a modulator of the UPR components. The increase in protein trafficking and protection from apoptosis, observed in KLF6-silenced cells, are consistent with its role in extravillous trophoblast migration and differentiation.