Inhibitors of Mek1/2 and Gsk3β, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.
Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia-induced changes was studied by using neutrophil-like HL-60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL-60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL-60 cells incubated with HG and S100B. HL-60 cells cultured in HG and S100B exhibited a 1.8-fold increase in fMLP-induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE-ERK1/2 pathway.
Neutrophils from people with poorly controlled diabetes present a primed phenotype and secrete excessive superoxide. Phospholipase A2 (PLA2)-derived arachidonic acid (AA) activates the assembly of NADPH oxidase to generate superoxide anion. There is a gap in the current literature regarding which PLA2 isoform regulates NADPH oxidase activation. The aim of this study was to identify the PLA2 isoform involved in the regulation of superoxide generation in neutrophils and investigate if PLA2 mediates priming in response to pathologic hyperglycemia. Neutrophils were isolated from people with diabetes mellitus and healthy controls, and HL60 neutrophil-like cells were grown in hyperglycemic conditions. Incubating neutrophils with the Ca2+-independent PLA2 (iPLA2) inhibitor bromoenol lactone (BEL) completely suppressed fMLP-induced generation of superoxide. The nonspecific actions of BEL on phosphatidic acid phosphohydrolase-1, p47phox phosphorylation, and apoptosis were ruled out by specific assays. Small interfering RNA knockdown of iPLA2 inhibited superoxide generation by neutrophils. Neutrophils from people with poorly controlled diabetes and in vitro incubation of neutrophils with high glucose and the receptor for advanced glycation end products ligand S100B greatly enhanced superoxide generation compared with controls, and this was significantly inhibited by BEL. A modified iPLA2 assay, Western blotting, and PCR confirmed that there was increased iPLA2 activity and expression in neutrophils from people with diabetes. AA (10 μM) partly rescued the inhibition of superoxide generation mediated by BEL, confirming that NADPH oxidase activity is, in part, regulated by AA. This study provides evidence for the role of iPLA2 in enhanced superoxide generation in neutrophils from people with diabetes mellitus and presents an alternate pathway independent of protein kinase C and phosphatidic acid phosphohydrolase-1 hydrolase signaling.
Lactoferrin accelerates bone formation, but the precise cellular mechanism behind this is still unclear. We examined the effect of lactoferrin on the differentiation of pluripotent mesenchymal cells using a typical pluripotent mesenchymal cell line, C2C12. Cells were cultured in low-mitogen differentiation medium to induce cell differentiation, with or without the addition of lactoferrin. The cell lineage was determined by alkaline phosphatase (ALPase) activity, mRNA expression of cellular phenotype-specific markers using real-time polymerase chain reaction (PCR), and protein synthesis using Western blotting. The expression of low-density lipoprotein lipase receptor-related proteins (LRPs) 1 and 2, both lactoferrin receptors, was determined by reverse transcription-PCR. ALPase activity increased after the addition of lactoferrin. The mRNA expression of Runx2, osteocalcin, and Sox9 increased markedly as a result of lactoferrin treatment, whereas the expression of MyoD, desmin, and PPARgamma decreased significantly. Western blots showed that lactoferrin stimulation increased Runx2 and Sox9 proteins, whereas it decreased MyoD and PPARgamma synthesis. C2C12 cells expressed the LRP1 lactoferrin receptor. These results indicate that lactoferrin treatment converts the differentiation pathway of C2C12 cells into the osteoblastic and chondroblastic lineage.
De novo establishment of DNA methylation is accomplished by DNMT3A and DNMT3B. Here, we analyze de novo DNA methylation in mouse embryonic fibroblasts (2i-MEFs) derived from DNA-hypomethylated 2i/L ES cells with genetic ablation of Dnmt3a or Dnmt3b. We identify 355 and 333 uniquely unmethylated genes in Dnmt3a and Dnmt3b knockout (KO) 2i-MEFs, respectively. We find that Dnmt3a is exclusively required for de novo methylation at both TSS regions and gene bodies of Polycomb group (PcG) target developmental genes, while Dnmt3b has a dominant role on the X chromosome. Consistent with this, tissue-specific DNA methylation at PcG target genes is substantially reduced in Dnmt3a KO embryos. Finally, we find that human patients with DNMT3 mutations exhibit reduced DNA methylation at regions that are hypomethylated in Dnmt3 KO 2i-MEFs. In conclusion, here we report a set of unique de novo DNA methylation target sites for both DNMT3 enzymes during mammalian development that overlap with hypomethylated sites in human patients.
Summary CpG islands (CGIs) including those at imprinting control regions (ICRs) are protected from de novo methylation in somatic cells. However, many cancers often exhibit CGI hypermethylation, implying that the machinery is impaired in cancer cells. Here, we conducted a comprehensive analysis of CGI methylation during somatic cell reprogramming. Although most CGIs remain hypomethylated, a small subset of CGIs, particularly at several ICRs, was often de novo methylated in reprogrammed pluripotent stem cells (PSCs). Such de novo ICR methylation was linked with the silencing of reprogramming factors, which occurs at a late stage of reprogramming. The ICR-preferred CGI hypermethylation was similarly observed in human PSCs. Mechanistically, ablation of Dnmt3a prevented PSCs from de novo ICR methylation. Notably, the ICR-preferred CGI hypermethylation was observed in pediatric cancers, while adult cancers exhibit genome-wide CGI hypermethylation. These results may have important implications in the pathogenesis of pediatric cancers and the application of PSCs.
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