Human Myeloid U937 Cells Research Articles

Differential expression and sorting of exosomal microRNAs upon activation of the human monocyte-like cell line U937

Extracellular vesicles such as exosomes in eukaryotes have drawn scrutiny due to their various roles in intercellular communication. Small RNAs, including microRNAs (miRNAs), are more abundant among the cargo of exosomes than other RNA types. MiRNAs loaded in secreted exosomes (or extracellular microRNAs) can be transported to recipient cells and may play a regulatory role although the miRNA loading (or sorting) mechanism in exosomes has not been clearly elucidated. Therefore, this study analyzed exosomal miRNA sequencing data from human myeloid U937 cells treated with phorbol 12-myristate 13-acetate (PMA) and compared it with data from PMA-untreated U937 cells. MiR-24 was highly expressed in the cytoplasm and exosomes of PMA-treated U937 cells. Also, miRNA pull-down and mass spectrophotometry analysis of PMA-treated U937 cells revealed that miR-24 was specifically associated with α-tubulin and hnRNP-E1 proteins. Furthermore, exosomal miR-24 was dramatically reduced when those proteins were inactivated with siRNAs, whereas cellular miR-24 showed no significant effect. We conclude that miR-24 is transported into exosomes from activated macrophages with the support of α-tubulin and hnRNP-E1.

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis. This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Analysis of SAMHD1 Restriction by Flow Cytometry in Human Myeloid U937 Cells.

Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis. This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.

Further Highlighting on the Prevention of Oxidative Damage by Polyphenol-Rich Wine Extracts.

Wine contains various polyphenols such as flavonoids, anthocyanins, and tannins. These molecules are responsible for the quality of wines, influencing their astringency, bitterness, and color and they are considered to have antioxidant activity. Polyphenols, extracted from grapes during the processes of vinification, could protect the body cells against reactive oxygen species level increase and could be useful to rescue several pathologies where oxidative stress represents the main cause. For that, in this study, red and white wine, provided by an Italian vinery (Marche region), have been analyzed. Chromatographic and morphofunctional analyses have been carried out for polyphenol extraction and to evaluate their protective effect on human myeloid U937 cells exposed to hydrogen peroxide. Both types of wines contained a mix of phenolic compounds with antioxidant properties and their content decreased, as expected, in white wine. Ultrastructural observations evidenced that wines, in particular red wine, strongly prevent mitochondrial damage and apoptotic cell death. In conclusion, the considered extracts show a relevant polyphenol content with strong antioxidant properties and abilities to prevent apoptosis. These findings suggest, for these compounds, a potential role in all pathological conditions where the body antioxidant system is overwhelmed.

Prospective multicentre study of the U-SENS test method for skin sensitization testing

The U-SENS™ is a test method based on the human myeloid U937 cell line to assess the skin sensitisation potential of substances. To demonstrate its robustness, a multicentre validation study with four laboratories testing 24 coded substances has been conducted according to internationally agreed principles. The primary objective of the study was to enlarge the U-SENS™'s reproducibility database. Secondary objectives were to provide additional evidence on its transferability and its predictive capability.Reproducibility within laboratories was approximately 92%, while the reproducibility between laboratories was 87.5%. Predictivity for the 24 validation substances was high, with sensitivity, specificity and accuracy being on average at least 93.8%. Similar performances are obtained for 38 substances when combining the study results with those of an earlier multicentre study, as well as with an automated version of the U-SENS™.With reliability and relevance similar to comparable non-animal skin sensitisation test methods, which have achieved regulatory acceptance, it is concluded that the U-SENS™ is a well reproducible and predictive test method. This profiles the U-SENS™ as a valuable addition to the suite of non-animal testing methods for skin sensitisation with the potential to significantly contribute to the development of integrated testing strategies.

Regulation of Human Formyl Peptide Receptor 1 Synthesis: Role of Single Nucleotide Polymorphisms, Transcription Factors, and Inflammatory Mediators

The gene encoding the human formyl peptide receptor 1 (FPR1) is heterogeneous, containing numerous single nucleotide polymorphisms (SNPs). Here, we examine the effect of these SNPs on gene transcription and protein translation. We also identify gene promoter sequences and putative FPR1 transcription factors. To test the effect of codon bias and codon pair bias on FPR1 expression, four FPR1 genetic variants were expressed in human myeloid U937 cells fused to a reporter gene encoding firefly luciferase. No significant differences in luciferase activity were detected, suggesting that the translational regulation and protein stability of FPR1 are modulated by factors other than the SNP codon bias and the variant amino acid properties. Deletion and mutagenesis analysis of the FPR1 promoter showed that a CCAAT box is not required for gene transcription. A −88/41 promoter construct resulted in the strongest transcriptional activity, whereas a −72/41 construct showed large reduction in activity. The region between −88 and −72 contains a consensus binding site for the transcription factor PU.1. Mutagenesis of this site caused significant reduction in reporter gene expression. The PU.1 binding was confirmed in vivo by chromatin immunoprecipitation, and the binding to nucleotides −84 to −76 (TTCCTATTT) was confirmed in vitro by an electrophoretic mobility shift assay. Thus, similar to many other myeloid genes, FPR1 promoter activity requires PU.1. Two single nucleotide polymorphisms at −56 and −54 did not significantly affect FPR1 gene expression, despite differences in binding of transcription factor IRF1 in vitro. Inflammatory mediators such as interferon-γ, tumor necrosis factor-α, and lipopolysaccharide did not increase FPR1 promoter activity in myeloid cells, whereas differentiation induced by DMSO and retinoic acid enhanced the activity. This implies that the expression of FPR1 in myeloid cells is developmentally regulated, and that the differentiated cells are equipped for immediate response to microbial infections.

Tetracycline Regulator Expression Alters the Transcriptional Program of Mammalian Cells

BackgroundTetracycline regulated ectopic gene expression is a widely used tool to study gene function. However, the tetracycline regulator (tetR) itself has been reported to cause certain phenotypic changes in mammalian cells. We, therefore, asked whether human myeloid U937 cells expressing the tetR in an autoregulated manner would exhibit alterations in gene expression upon removal of tetracycline.Methodology/Principal FindingsMicroarray analyses revealed that 172 and 774 unique genes were significantly differentially expressed by at least 2- or 1.5-fold, respectively, when tetR expressing U937 cells were maintained in media with or without the antibiotic.Conclusions/SignificanceThese alterations in gene expression are likely to contribute to the phenotypic consequences of tetR expression. In addition, they need to be taken into consideration when using the tetR system for the identification of target genes of transcription factors or other genes of interest.

2P-225 MHz超音波によるU937細胞におけるシグナルトランスダクションの発現(放射線生物-活性酸素,第47回日本生物物理学会年会)

2P-225 MHz超音波によるU937細胞におけるシグナルトランスダクションの発現(放射線生物-活性酸素,第47回日本生物物理学会年会)

C/EBPϵ interacts with retinoblastoma and E2F1 during granulopoiesis

AbstractCCAAT enhancer binding protein epsilon (C/EBPϵ) is a myeloid specific transcription factor that is essential for terminal granulocytic differentiation. Retinoblastoma (Rb) and E2F1 are critical cell cycle regulators that also have been implicated in several differentiation systems. Here, we demonstrate that C/EBPϵ interacts with Rb and E2F1 during granulocytic differentiation in NB4 and U937 human myeloid cells and in 32Dcl3 murine myeloid precursor cells. The interaction between C/EBPϵ and Rb enhances C/EBPϵ-mediated transcription of myeloid specific genes both in reporter assays and endogenously. The C/EBPϵ-E2F1 interaction results in repression of E2F1-mediated transcriptional activity. Finally, overexpression of C/EBPϵ in human myeloid cells leads to down-regulation of c-Myc. We propose that the interactions between C/EBPϵ, a tissue-specific transcription factor, and the broad-spectrum proteins, Rb and E2F1, are important in C/EBPϵ-induced terminal granulocytic differentiation.

GAMMA-INTERFERON (IFN-γ) AUGMENTS APOPTOTIC RESPONSE TO MISTLETOE LECTIN-II VIA UPREGULATION OF FAS/FAS L EXPRESSION AND CASPASE ACTIVATION IN HUMAN MYELOID U937 CELLS

Mistletoe lectin-II, a major composition of Korean mistletoe (Viscum album coloratum), is known as a potent apoptosis inducer. The previous research has demonstrated that Korean mistletoe lectin-II induces apoptosis via c-Jun N terminal kinase (JNK) activation in human myeloid U937 cells. The purpose of this research is to prove the synergistic action of mistletoe lectin-II and interferon-γ (IFN-γ) in the apoptotic cytotoxicity of U937. When U937 cells were treated with mistletoe lectin-II after being differentiated by IFN-γ, the proteolytic activity of caspase-3 and 9 was markedly elevated and that of caspase-8 was prolonged for 18 hr. The activation of caspase-3-like protease requires the earlier cleavage of poly(ADP-ribose) polymerase(PARP). Caspase-1 was, however, not activated during the resting phase and nor in IFN-γ-differentiated U937 cells. Western blot analysis revealed that, in IFN-γ-differentiated U937 cells, the expression of Fas (CD95/APO-1) & Fas ligand(FasL) increases the apoptotic sensitivity against Mistletoe lectin-II. Fas (CD95/APO-1) & FasL were not significantly induced solely by mistletoe lectin-II. Furthermore the activity of JNK1 in U937 cells was also markedly increased with IFN-γ-differentiation, compared to that of the control. These results suggest that the IFN-γ-differentiation of U937 cells increases the susceptibility to mistletoe lectin-II-induced apoptosis.

Characterization and localization to chromosome 7 of psihGABPalpha, a human processed pseudogene related to the ets transcription factor, hGABPalpha.

GABP is a heteromeric transcription factor complex which consists of the ets related protein, GABPalpha, and the Notch-related protein, GABPbeta. We isolated a human genomic DNA fragment which is highly homologous and colinear with human GABPalpha cDNA, but which lacks introns. This processed pseudogene, psihGABPalpha, is expressed as RNA in U937 human myeloid cells, but a mutation at the site that corresponds to the ATG start methionine codon prevents its translation into protein. The pseudogene was localized to chromosome 7 using a somatic cell hybrid mapping panel and it is not syntenic with authentic GABPalpha, which was localized to chromosome 21. We have identified psihGABPalpha, a novel, GABPalpha-related processed pseudogene which is expressed as a RNA transcript in human myeloid cells.

Identification and Characterization of a Novel Cytokine, THANK, aTNF Homologue That ActivatesApoptosis, Nuclear Factor-κB, and c-Jun NH2-Terminal Kinase

By using the amino acid sequence motif of tumor necrosis factor (TNF), we searched the expressed sequence tag data base and identified a novel full-length cDNA encoding 285 amino acid residues and named it THANK. THANK is a type II transmembrane protein with 15-20% overall amino acid sequence homology to TNF, LT-alpha, FasL, and LIGHT, all members of the TNF family. The mRNA for THANK was expressed at high levels by peripheral blood leukocytes, lymph node, spleen, and thymus and at low levels by small intestine, pancreas, placenta, and lungs. THANK was also prominently expressed in hematopoietic cell lines. The recombinant purified protein expressed in the baculovirus system had an approximate molecular size 20 kDa with amino-terminal sequence of AVQGP. Treatment of human myeloid U937 cells with purified THANK activated nuclear transcription factor-kappaB (NF-kappaB) consisting of p50 and p65. Activation was time- and dose-dependent, beginning with as little as a 1 pM amount of the cytokines and as early as 15 min. Under the same conditions, THANK also activated c-jun NH2-terminal kinase (JNK) in U937 cells. THANK also strongly suppressed the growth of tumor cell lines and activated caspase-3. Although THANK had all the activities and potency of TNF, it did not bind to the TNF receptors. Thus our results indicate that THANK is a novel cytokine that belongs to the TNF family and activates apoptosis, NF-kappaB, and JNK through a distinct receptor.

Suppression of tumor necrosis factor-activated nuclear transcription factor-κB, activator protein-1, c-Jun N-terminal kinase, and apoptosis by β-lapachone

β-Lapachone, the product of a tree from South America, is known to exhibit various pharmacologic properties, the mechanisms of which are poorly understood. In the present report, we examined the effect of β-lapachone on the tumor necrosis factor (TNF)-induced activation of the nuclear transcription factors NF-κB and activator protein-1 (AP-1) in human myeloid U937 cells. TNF-induced NF-κB activation, p65 translocation, IκBα degradation, and NF-κB-dependent reporter gene expression were inhibited in cells pretreated with β-lapachone. Direct treatment of the p50-p65 heterodimer of NF-κB with β-lapachone had no effect on its ability to bind to the DNA. Besides myeloid cells, β-lapachone was also inhibitory in T-cells and epithelial cells. β-Lapachone also suppressed the activation of NF-κB by lipopolysaccharide, okadaic acid, and ceramide but had no significant effect on activation by H 2O 2 or phorbol myristate acetate, indicating that its action is selective. β-Lapachone also abolished TNF-induced activation of AP-1, c-Jun N-terminal kinase, and mitogen-activated protein kinase kinase (MAPKK or MEK). TNF-induced cytotoxicity and activation of caspase-3 were also abolished by β-lapachone. Because reducing agents (dithiothreitol and N-acetylcysteine) reversed the effect of β-lapachone, it suggests the role of a critical sulfhydryl group. Overall, our results identify NF-κB, AP-1, and apoptosis as novel targets for β-lapachone, and this may explain some of its pharmacologic effects.

Multiple activation steps of the N-formyl peptide receptor.

The human N-formyl peptide receptor (FPR) is representative of a growing family of G protein-coupled receptors (GPCR) that respond to chemokines and chemoattractants. Despite the importance of this receptor class to immune function, relatively little is known about the molecular mechanisms involved in their activation. To reveal steps required for the activation of GPCR receptors, we utilized mutants of the FPR which have previously been shown to be incapable of binding and activating G proteins. For this study, the FPR mutants were expressed in human myeloid U937 cells and characterized for functions in addition to G protein coupling, such as receptor phosphorylation and ligand-induced receptor internalization. The results demonstrated that one of the mutants, R123G, though being unable to activate G protein, was capable of undergoing ligand-induced phosphorylation as well as internalization. Receptor internalization was monitored by following the fate of the ligand as well as by directly monitoring the fate of the receptor. The results with the R123G mutant were in contrast to those obtained for mutants D71A and R309G/E310A/R311G which, though being expressed at the cell surface and binding ligand, were incapable of being phosphorylated or internalized upon agonist stimulation. These results suggest that following ligand binding at least two "steps" are required for full activation of the wild-type FPR. That these observations may be of more general importance in GPCR-mediated signaling is suggested by the highly conserved nature of the mutants studied: D71, R123, and the site represented by amino acids 309-311 are very highly conserved throughout the entire superfamily of G protein-coupled receptors. Models of receptor activation based on the observed results are discussed.

Protein Tyrosine Kinase Inhibitors Block Tumor Necrosis Factor-Induced Activation of Nuclear Factor-κB, Degradation of IκBα, Nuclear Translocation of p65, and Subsequent Gene Expression

Several inflammatory effects of tumor necrosis factor (TNF) are known to be mediated through activation of a nuclear transcription factor NF-κB, but how TNF activates NF-κB is incompletely understood. In the present report, we examined the role of protein tyrosine kinases (PTK) in TNF-mediated NF-κB activation by using genistein and erbstatin, two potent inhibitors of PTK. The treatment of human myeloid U-937 cells with either inhibitor completely suppressed the TNF-induced NF-κB activation in a dose- and time-dependent manner. Suppression correlated with PTK activity, since among the structural analogues of genistein, only an active inhibitor of PTK, quercetin blocked TNF-induced NF-κB activation and not daidzein, an inactive inhibitor. Inhibition of NF-κB activation was not limited to myeloid cells, as it was observed with T cells and epithelial cells. Both the PTK inhibitors blocked the degradation of IκBα, the inhibitory subunit of NF-κB, and the consequent translocation of the p65 subunit without any significant effect on p50 or on c-Rel. The PTK inhibitors did not interfere with NF-κB binding to DNA. The NF-κB-dependent CAT reporter gene expression in transient transfection assays was also suppressed by the PTK inhibitors. Both PTK inhibitors abolished TNF-induced activation of N-terminal c-Jun kinase and mitogen-activated protein kinase kinase. Overall, our results suggest that a genistein- and erbstatin-sensitive PTK is involved in the pathway leading to NF-κB activation and gene expression by TNF and thus could be used as a target for development of antiinflammatory drugs.

Transcription of the Human Colony-Stimulating Factor-1 Receptor Gene Is Regulated by Separate Tissue-Specific Promoters

Receptors for macrophage colony-stimulating factor (CSF-1R) are expressed not only by monocytes, macrophages, and their progenitors, but also by placental trophoblasts during fetal development. In monocytes, CSF-1R gene transcripts originate at multiple sites immediately upstream of the gene's coding sequences, whereas in placental cells the transcripts include an additional noncoding exon, located 26 kb upstream near the 3' end of the B-type platelet-derived growth factor (PDGF) receptor gene. Physically distinct CSF-1R transcription origins suggest separate promoter usage by the two cell types. To identify regulatory elements of these promoters, we fused CSF-1R genomic sequences to bacterial reporter genes and introduced the resulting constructs into human cell lines and mouse fibroblasts. A 775-bp genomic fragment containing CSF-1R placental transcription origins and adjacent upstream sequences mediated reporter gene expression in BeWo choriocarcinoma cells and mouse NIH-3T3 fibroblasts, but not in myeloid or lymphoid cells. By contrast, a 550-bp genomic fragment containing CSF-1R monocyte transcription origins and 5' flanking sequences directed gene expression in U-937 human myeloid cells, but not in the other cell types. Thus, nucleotide sequences of fewer than 1,000-bp upstream of the two independent CSF-1R transcription origins contain the minimal promoter elements needed to program appropriate tissue-specific expression of reporter genes.

Transcription of the human colony-stimulating factor-1 receptor gene is regulated by separate tissue-specific promoters

Receptors for macrophage colony-stimulating factor (CSF-1R) are expressed not only by monocytes, macrophages, and their progenitors, but also by placental trophoblasts during fetal development. In monocytes, CSF-1R gene transcripts originate at multiple sites immediately upstream of the gene's coding sequences, whereas in placental cells the transcripts include an additional noncoding exon, located 26 kb upstream near the 3′ end of the B-type platelet-derived growth factor (PDGF) receptor gene. Physically distinct CSF-1R transcription origins suggest separate promoter usage by the two cell types. To identify regulatory elements of these promoters, we fused CSF- 1R genomic sequences to bacterial reporter genes and introduced the resulting constructs into human cell lines and mouse fibroblasts. A 775- bp genomic fragment containing CSF-1R placental transcription origins and adjacent upstream sequences mediated reporter gene expression in BeWo choriocarcinoma cells and mouse NIH-3T3 fibroblasts, but not in myeloid or lymphoid cells. By contrast, a 550-bp genomic fragment containing CSF-1R monocyte transcription origins and 5′ flanking sequences directed gene expression in U-937 human myeloid cells, but not in the other cell types. Thus, nucleotide sequences of fewer than 1,000-bp upstream of the two independent CSF-1R transcription origins contain the minimal promoter elements needed to program appropriate tissue-specific expression of reporter genes.