KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis
KLF6 Induces Apoptosis in Human Lens Epithelial Cells Through the ATF4-ATF3-CHOP Axis
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DOI：
10.2147/dddt.s218467
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发表时间：
2020-01-01
影响因子：
4.8
通讯作者：
Dong, Lijie
中科院分区：
文献类型：
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作者：
Tian, Fang;Zhao, Jinzhi;Dong, Lijie
背景：大量研究证实高度近视与白内障的高发病率有关，白内障的发生是由于内质网应激引起晶状体上皮细胞(LECs)的凋亡所致。Kruppel-like factor6(KLF6)是一种肿瘤抑制因子，参与细胞增殖和凋亡的调节。目的：探讨KLF6诱导晶状体上皮细胞凋亡与ATF4(激活转录因子4)-ATF3(激活转录因子3)-CHOP(C/EBP同源蛋白)信号通路的关系。用小干扰RNA(SiRNA)进行ATF4、ATF3和CHOP基因敲除。QT-PCR和Western-blotting检测相关基因的表达。结果：异位表达ATF4、ATF3和CHOP可阻断KLF6诱导的细胞凋亡，免疫印迹法检测细胞凋亡标志物Bax和Bcl2的表达，DCFH-DA探针检测细胞内ROS的表达水平。此外，我们还发现ATF4可以调节ATF3和CHOP的表达，并且ATF3的沉默可以在不改变ATF4水平的情况下减少CHOP的表达；然而，阻断CHOP并不影响ATF4和ATF3的表达，这表明KLF6通过介导ATF4-ATF3/CHOP轴来触发晶状体上皮细胞的内质网应激。结论：KLF6过表达在KLF6诱导HLECs凋亡中起重要作用。我们的结果增加了我们对LEC凋亡调控机制的理解，并有助于开发一种新的白内障预防策略。
Background: Many studies have confirmed that high myopia is related to the high prevalence of cataracts, which results from apoptosis of lens epithelial cells (LECs) due to endoplasmic reticulum stress. Kruppel-like factor 6 (KLF6) is a tumor suppressor that is involved in the regulation of cell proliferation and apoptosis.Purpose: In this study, our purpose was to find the relationship between KLF6-induced apoptosis in LECs and ATF4 (activating transcription factor 4)-ATF3 (activating transcription factor 3)-CHOP (C/EBP homologous protein) signaling pathway.Methods: KLF6, ATF4, ATF3, and CHOP were ectopically expressed using cDNAs sub-cloned into the pCDNA3.1+ vector. ATF4, ATF3, and CHOP knockdown were performed by small interfering RNA (siRNA). Expression of relative gene was tested using QT-PCR and western-blot. Then, accompanied by UVB stimulation, cell viability was measured by CCK-8 assay; The cell damage was examined by live & dead staining; The apoptotic markers Bax and Bcl-2 were detected by immunoblotting; Quantitative apoptotic levels were measured with the Apoptosis Detection Kit; The expression level of reactive oxygen-free radical (ROS) was analyzed by DCFH-DA probe.Results: Ectopically expressed ATF4, ATF3, and CHOP-induced apoptosis in cells, whereas ATF4, ATF3, and CHOP knockdown by small interfering RNA (siRNA) blocked KLF6-induced apoptosis. In addition, we determined that ATF4 regulates ATF3 and CHOP expression and that ATF3 silencing reduces CHOP upregulation without changing ATF4 levels; however, ATF4 and ATF3 expression was unaffected by blockade of CHOP, suggesting that KLF6 triggers endoplasmic reticulum stress in LECs by mediating the ATF4-ATF3/CHOP axis. Besides, KLF6 overexpression significantly induced LEC apoptosis under UV radiation, as demonstrated by the elevated Bax/Bcl-2 ratio.Conclusion: The ATF4-ATF3-CHOP pathway plays an important role in KLF6-induced apoptosis in HLECs. Our results increase our understanding of the mechanisms that regulate LEC apoptosis and contribute to the development of a new preventative strategy for cataract.