Binó Lucia, Kučera Jan, Štefková Kateřina, Švihálková Šindlerová Lenka, Lánová Martina, Kudová Jana, Kubala Lukáš, Pacherník Jiří
Institute of Biophysics, Academy of Sciences of the Czech Republic v. v. i., Královopolská 135, 612 65 Brno, Czech Republic; Institute of Experimental Biology, Department of Physiology and Immunology of Animals, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic; Department of Histology and Embryology, Faculty of Medicine, Masaryk University, Brno, Czech Republic.
Institute of Experimental Biology, Department of Physiology and Immunology of Animals, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic.
Chem Biol Interact. 2016 Jan 25;244:204-14. doi: 10.1016/j.cbi.2015.12.007. Epub 2015 Dec 23.
Hypoxic conditions are suggested to affect the differentiation status of stem cells (SC), including embryonic stem cells (ESC). Hypoxia inducible factor (HIF) is one of the main intracellular molecules responsible for the cellular response to hypoxia. Hypoxia stabilizes HIF by inhibiting the activity of HIF prolyl-hydroxylases (PHD), which are responsible for targeting HIF-alpha subunits for proteosomal degradation. To address the impact of HIF stabilization on the maintenance of the stemness signature of mouse ESC (mESC), we tested the influence of the inhibition of PHDs and hypoxia (1% O2 and 5% O2) on spontaneous ESC differentiation triggered by leukemia inhibitory factor withdrawal for 24 and 48 h. The widely used panhydroxylase inhibitor dimethyloxaloylglycine (DMOG) and PHD inhibitor JNJ-42041935 (JNJ) with suggested higher specificity towards PHDs were employed. Both inhibitors and both levels of hypoxia significantly increased HIF-1alpha and HIF-2alpha protein levels and HIF transcriptional activity in spontaneously differentiating mESC. This was accompanied by significant downregulation of cell proliferation manifested by the complete inhibition of DNA synthesis and partial arrest in the S phase after 48 h. Further, HIF stabilization enhanced downregulation of the expressions of some pluripotency markers (OCT-4, NANOG, ZFP-42, TNAP) in spontaneously differentiating mESC. However, at the same time, there was also a significant decrease in the expression of some genes selected as markers of cell differentiation (e.g. SOX1, BRACH T, ELF5). In conclusion, the short term stabilization of HIF mediated by the PHD inhibitors JNJ and DMOG and hypoxia did not prevent the spontaneous loss of pluripotency markers in mESC. However, it significantly downregulated the proliferation of these cells.
低氧条件被认为会影响干细胞（SC）的分化状态，包括胚胎干细胞（ESC）。缺氧诱导因子（HIF）是细胞对缺氧反应的主要细胞内分子之一。低氧通过抑制HIF脯氨酰羟化酶（PHD）的活性来稳定HIF，PHD负责将HIF-α亚基靶向蛋白酶体降解。为了研究HIF稳定对小鼠胚胎干细胞（mESC）干性特征维持的影响，我们测试了抑制PHD和低氧（1% O₂和5% O₂）对白血病抑制因子撤除24小时和48小时引发的mESC自发分化的影响。使用了广泛应用的泛羟化酶抑制剂二甲基草酰甘氨酸（DMOG）和对PHD具有更高特异性的PHD抑制剂JNJ-42041935（JNJ）。两种抑制剂以及两种低氧水平均显著增加了自发分化的mESC中HIF-1α和HIF-2α蛋白水平以及HIF转录活性。这伴随着细胞增殖的显著下调，表现为DNA合成完全抑制以及48小时后S期部分停滞。此外，HIF稳定增强了自发分化的mESC中一些多能性标志物（OCT-4、NANOG、ZFP-42、TNAP）表达的下调。然而，与此同时，一些被选为细胞分化标志物的基因（如SOX1、BRACH T、ELF5）的表达也显著降低。总之，由PHD抑制剂JNJ和DMOG以及低氧介导的HIF短期稳定并不能阻止mESC中多能性标志物的自发丧失。然而，它显著下调了这些细胞的增殖。
