The histone demethylase JMJD2C constitutes a novel NFE2 target gene that is required for the survival of JAK2V617F mutated cells.

The transcription factor NFE2 is overexpressed in most patients with myeloproliferative neoplasms (MPN). Moreover, mutations in NFE2, found in a subset of MPN patients, strongly predispose for transformation to acute leukemia. Transgenic mice overexpressing NFE2 as well as mice harboring NFE2 mutations display an MPN phenotype and spontaneously develop leukemia. However, the molecular mechanisms effecting NFE2-driven leukemic transformation remain incompletely understood. Here we show that the pro-leukemic histone demethylase JMJD2C constitutes a novel NFE2 target gene. JMJD2C expression is elevated in MPN patients as well as in NFE2 transgenic mice. Moreover, we show that loss of JMJD2C selectively impairs proliferation of JAK2V617F mutated cells. Our data suggest that JMJD2C represents a promising drug target in MPN and provide a rationale for further investigation in preclinical and clinical settings.

CEACAM3 decreases asthma exacerbations and modulates respiratory syncytial virus latent infection in children.

BACKGROUND: Respiratory syncytial virus (RSV) is associated with childhood asthma. Nevertheless, not all children exposed to RSV develop asthma symptoms, possibly because genes modulate the effects of RSV on asthma exacerbations. OBJECTIVE: The purpose of this study was to identify genes that modulate the effect of RSV latent infection on asthma exacerbations. METHODS: We performed a meta-analysis to investigate differentially expressed genes (DEGs) of RSV infection from Gene Expression Omnibus datasets. Expression quantitative trait loci (eQTL) methods were applied to select single nucleotide polymorphisms (SNPs) that were associated with DEGs. Gene-based analysis was used to identify SNPs that were significantly associated with asthma exacerbations in the Taiwanese Consortium of Childhood Asthma Study (TCCAS), and validation was attempted in an independent cohort, the Childhood Asthma Management Program (CAMP). Gene-RSV interaction analyses were performed to investigate the association between the interaction of SNPs and RSV latent infection on asthma exacerbations. RESULTS: A total of 352 significant DEGs were found by meta-analysis of RSV-related genes. We used 38 123 SNPs related to DEGs to investigate the genetic main effects on asthma exacerbations. We found that eight RSV-related genes (GADD45A, GYPB, MS4A3, NFE2, RNASE3, EPB41L3, CEACAM6 and CEACAM3) were significantly associated with asthma exacerbations in TCCAS and also validated in CAMP. In TCCAS, rs7251960 (CEACAM3) significantly modulated the effect of RSV latent infection on asthma exacerbations (false-discovery rate <0.05). The rs7251960 variant was associated with CEACAM3 mRNA expression in lung tissue (p for trend=1.2×10-7). CEACAM3 mRNA was reduced in nasal mucosa from subjects with asthma exacerbations in two independent datasets. CONCLUSIONS: rs7251960 is an eQTL for CEACAM3, and CEACAM3 mRNA expression is reduced in subjects experiencing asthma exacerbations. CEACAM3 may be a modulator of RSV latent infection on asthma exacerbations.

Nuclear factor erythroid 2 (NF-E2) p45-related factor 2 interferes with homeodomain-interacting protein kinase 2/p53 activity to impair solid tumors chemosensitivity.

Resistance to chemotherapy represents a major hurdle to successful cancer treatment. A key role for efficient response to anticancer therapies is played by TP53 oncosuppressor gene that indeed is mutated in 50% of human cancers or inactivated at protein level in the remaining 50%. Homeodomain-interacting protein kinase 2 (HIPK2) is the wild-type p53 (wtp53) apoptotic activator, and its inhibition by hypoxia or hyperglycemia may contribute to tumor chemoresistance mainly by impairing p53 apoptotic activity. Another important molecule able to induce chemoresistance is nuclear factor erythroid 2 (NF-E2) p45-related factor 2 (NRF2) transcription factor, whose activation by oxidative and/or electrophilic stress regulates a transcriptional antioxidant program allowing cancer cells to adapt and survive to stresses. NRF2 may shift from cytoprotective to tumor-promoting function, according to tumor phases. NRF2 may crosstalk with both wtp53 and mutant p53 (mutp53), inhibiting the wtp53 apoptotic function and strengthening the mutp53 oncogenic function. NRF2 has also been shown to induce HIPK2 mRNA expression cooperating in inducing cytoprotection. Although HIPK2, p53, and NRF2 have been individually extensively studied, their interplay has not been clearly addressed yet. On the basis of the background and our results, we aim at hypothesizing the unexpected pro-survival activity played by the NRF2/HIPK2/p53 interplay that can be hijacked by cancer cells to bypass drugs cytotoxicity.

The transcription factor NFE2 enhances expression of the hematopoietic master regulators SCL/TAL1 and GATA2.

Activity of the transcription factor NFE2 is elevated in the majority of patients with myeloproliferative neoplasms (MPNs), either by overexpression of the wild-type alleles or by the presence of an activating mutation. In murine models, enhanced NFE2 activity causes an MPN phenotype with spontaneous transformation to acute leukemia. However, little is known about the downstream target genes activated by augmented NFE2 levels. Here, we describe that NFE2 regulates expression of the hematopoietic master regulators GATA2 and SCL/TAL1, which are in turn overexpressed in primary MPN cells, suggesting that concomitant aberrant activation of several transcription factors coordinately contributes to the cellular expansion characteristic of these disorders.

Loss of NF-E2 expression contributes to the induction of profibrotic signaling in diabetic kidneys.

AIMS: This study aimed to examine the anti-fibrotic role of Nuclear Factor-Erythroid derived 2 (NF-E2) in human renal tubule (HK-11) cells and in type 1 and type 2 diabetic (T1D, T2D) mouse kidneys. MAIN METHODS: Anti-fibrotic effects of NF-E2 were examined in transforming growth factor-ß (TGF-ß) treated HK-11 cells by over-expressing/silencing NF-E2 expression and determining its effects on profibrotic signaling. NF-E2 proteasomal degradation was confirmed by proteasome inhibition in HK-11 cells and diabetic mice. Clinical relevance of changes in NF-E2 expression to fibrotic changes in the kidney were assessed in T1D and T2D mouse kidneys. KEY FINDINGS: NF-E2 expression was significantly decreased in TGF-ß treated HK-11 cells and in kidneys of diabetic mice with concurrent increase in expression of fibrotic proteins. TGF-ß treatment of HK-11 cells did not inhibit NF-E2 mRNA expression, suggesting that the post-translational changes may contribute to NF-E2 protein degradation. The down-regulation of NF-E2 expression was attributed to its proteasomal degradation, as TGF-ß- and diabetes-induced NF-E2 down regulation was prevented by proteasome inhibitor treatment. In HK-11 cells TGF-ß treatment decreased E-cadherin expression and induced pSer82Hsp27/NF-E2 association, likely to promote NF-E2 degradation, as Hsp27 can target proteins to the proteasome. A critical role for NF-E2 in regulation of renal fibrosis was demonstrated as over-expression of NF-E2 or silencing NF-E2 expression, decreased or increased profibrotic proteins in TGF-ß-treated HK-11 cells, respectively. SIGNIFICANCE: NF-E2, a novel anti-fibrotic protein, is down-regulated in diabetic kidneys. Preserving/inducing NF-E2 expression in diabetic kidneys may provide a therapeutic potential to combat DN.

GATA-1: A potential novel biomarker for the differentiation of essential thrombocythemia and myelofibrosis.

Essentials The BCR-ABL negative myeloproliferative neoplasms are subjected to unknown phenotypic modifiers. GATA-1 is upregulated in ET patients, regardless of treatment regimen or mutational status. Myelofibrosis (MF) megakaryocytes displayed decreased GATA-1 staining. GATA-1 may have utility as a diagnostic marker in ET and in its differential diagnosis from MF. ABSTRACT: Background The BCR-ABL-negative myeloproliferative neoplasms, i.e., polycythemia vera, essential thrombocythemia (ET), and myelofibrosis (MF), are characterized by mutations in JAK2, CALR, or MPL. However, an as yet unknown factor drives the precise disease phenotype. The hematopoietic transcription factor GATA-1 and its downstream targets NFE2 and FLI1 are responsible for determining erythroid and megakaryocyte lineages during hematopoietic stem cell differentiation. Previous studies have demonstrated a low level of GATA-1 expression in megakaryocytes from patients with MF. Objectives and methods The expression of GATA-1, NFE2 and FLI1 was studied for changes in the peripheral blood (PB) of ET patients. Peripheral blood samples were obtained from 36 ET patients, 14 MF patients, and seven healthy control donors. Total RNA from PB mononuclear cells (PBMCs) was extracted, and quantitative polymerase chain reaction was used to determine relative changes in gene expression. Protein levels of GATA-1 were also determined in bone marrow sections from ET and MF patients. Results GATA-1 mRNA was upregulated in ET patients, regardless of treatment regimen or mutational status. FLI1 expression was significantly downregulated, whereas NFE2 expression was unaffected by changes in GATA-1 mRNA levels. Megakaryocytes from ET patients showed increased protein levels of GATA-1 as compared with those from MF patients. Conclusions Our results confirmed, in PB, our previous data demonstrating elevated levels of GATA-1 mRNA in total bone marrow of ET patients. GATA-1 mRNA levels are independent of cytoreductive therapies, and may have utility as a diagnostic marker in ET and in its differential diagnosis from MF.

CD226 is involved in megakaryocyte activation and early-stage differentiation.

This study was conducted to investigate the effect of CD226 on the differentiation, activation, and polyploidization of megakaryocytes (MKs) and explore the potential mechanism. Dami (megakaryocyte line) cell maturation was induced by phorbol 12-myristate 13-acetate. CD226 was silenced by infection with a CD226-specific shRNA lentiviral vector. The mRNA level of CD226 was detected by qRT-PCR. The expressions of Dami cells surface CD226, MK specific markers CD41 and CD62P, and DNA ploidy in Dami cells and CD226 knockdown (KD) cells were evaluated by flow cytometry. The effect of CD226 on the expression of megakaryocyte-associated transcription factors was measured by western blot and confocal analysis. Transfection with CD226 shRNA lentivirus dramatically decreased the level of CD226 and expression of CD62 P in Dami cells. Silencing of CD226 caused morphological changes and differentiation retardation in low-ploidy MK. Furthermore, CD226 knockout (KO) mice exhibited increased 2N-4N low-ploidy MK and decreased ≥8N polyploidy. Interestingly, silencing of CD226 in megakaryocytic cells down-regulated the expression of early stage transcription factors includes GATA-binding factor 1 (GATA-1) and friend leukemia integration 1 (FLI-1), but not late-stage nuclear factor, erythroid 2 (NF-E2). CD226 is involved in MKs activation and polyploidy cell cycle control.

Altered NFE2 activity predisposes to leukemic transformation and myelosarcoma with AML-specific aberrations.

In acute myeloid leukemia (AML), acquired genetic aberrations carry prognostic implications and guide therapeutic decisions. Clinical algorithms have been improved by the incorporation of novel aberrations. Here, we report the presence and functional characterization of mutations in the transcription factor NFE2 in patients with AML and in a patient with myelosarcoma. We previously described NFE2 mutations in patients with myeloproliferative neoplasms and demonstrated that expression of mutant NFE2 in mice causes a myeloproliferative phenotype. Now, we show that, during follow-up, 34% of these mice transform to leukemia presenting with or without concomitant myelosarcomas, or develop isolated myelosarcomas. These myelosarcomas and leukemias acquired AML-specific alterations, including the murine equivalent of trisomy 8, loss of the AML commonly deleted region on chromosome 5q, and mutations in the tumor suppressor Trp53 Our data show that mutations in NFE2 predispose to the acquisition of secondary changes promoting the development of myelosarcoma and/or AML.

Loss of fibrinogen in zebrafish results in an asymptomatic embryonic hemostatic defect and synthetic lethality with thrombocytopenia.

Essentials Loss of fibrinogen in zebrafish has been previously shown to result in adult onset hemorrhage Hemostatic defects were discovered in early fga-/- embryos but well tolerated until adulthood Afibrinogenemia and thrombocytopenia results in synthetic lethality in zebrafish. Testing human FGA variants of uncertain significance in zebrafish identified causative mutations SUMMARY: Background Mutations in the alpha chain of fibrinogen (FGA), such as deficiencies in other fibrinogen subunits, lead to rare inherited autosomal recessive hemostatic disorders. These range from asymptomatic to catastrophic life-threatening bleeds and the molecular basis of inherited fibrinogen deficiencies is only partially understood. Zinc finger nucleases have been used to produce mutations in zebrafish fga, resulting in overt adult-onset hemorrhage and reduced survival. Objectives To determine the age of onset of hemostatic defects in afibrinogenemic zebrafish and model human fibrinogen deficiencies. Methods TALEN genome editing (transcription activator-like effector nucleases) was used to generate a zebrafish fga mutant. Hemostatic defects were assessed through survival, gross anatomical and histological observation and laser-induced endothelial injury. Human FGA variants with unknown pathologies were engineered into the orthologous positions in zebrafish fga. Results Loss of Fga decreased survival and resulted in synthetic lethality when combined with thrombocytopenia. Zebrafish fga mutants exhibit a severe hemostatic defect by 3 days of life, but without visible hemorrhage. Induced thrombus formation through venous endothelial injury was completely absent in mutant embryos and larvae. This hemostatic defect was restored by microinjection of wild-type fga cDNA plasmid or purified human fibrinogen. This system was used to determine whether unknown human variants were pathological by engineering them into fga. Conclusions These studies confirm that loss of fibrinogen in zebrafish results in the absence of hemostasis from the embryonic period through adulthood. When combined with thrombocytopenia, zebrafish exhibit synthetic lethality, demonstrating that thrombocytes are necessary for survival in response to hemorrhage.

Phenethyl Isothiocyanate, a Dual Activator of Transcription Factors NRF2 and HSF1.

Cruciferous vegetables are rich sources of glucosinolates which are the biogenic precursor molecules of isothiocyanates (ITCs). The relationship between the consumption of cruciferous vegetables and chemoprotection has been widely documented in epidemiological studies. Phenethyl isothiocyanate (PEITC) occurs as its glucosinolate precursor gluconasturtiin in the cruciferous vegetable watercress (Nasturtium officinale). PEITC has multiple biological effects, including activation of cytoprotective pathways, such as those mediated by the transcription factor nuclear factor erythroid 2 p45-related factor 2 (NRF2) and the transcription factor heat shock factor 1 (HSF1), and can cause changes in the epigenome. However, at high concentrations, PEITC leads to accumulation of reactive oxygen species and cytoskeletal changes, resulting in cytotoxicity. Underlying these activities is the sulfhydryl reactivity of PEITC with cysteine residues in its protein targets. This chemical reactivity highlights the critical importance of the dose of PEITC for achieving on-target selectivity, which should be carefully considered in the design of future clinical trials.

Epigenetic regulation of NFE2 overexpression in myeloproliferative neoplasms.

The transcription factor "nuclear factor erythroid 2" (NFE2) is overexpressed in the majority of patients with myeloproliferative neoplasms (MPNs). In murine models, elevated NFE2 levels cause an MPN phenotype with spontaneous leukemic transformation. However, both the molecular mechanisms leading to NFE2 overexpression and its downstream targets remain incompletely understood. Here, we show that the histone demethylase JMJD1C constitutes a novel NFE2 target gene. JMJD1C levels are significantly elevated in polycythemia vera (PV) and primary myelofibrosis patients; concomitantly, global H3K9me1 and H3K9me2 levels are significantly decreased. JMJD1C binding to the NFE2 promoter is increased in PV patients, decreasing both H3K9me2 levels and binding of the repressive heterochromatin protein-1α (HP1α). Hence, JMJD1C and NFE2 participate in a novel autoregulatory loop. Depleting JMJD1C expression significantly reduced cytokine-independent growth of an MPN cell line. Independently, NFE2 is regulated through the epigenetic JAK2 pathway by phosphorylation of H3Y41. This likewise inhibits HP1α binding. Treatment with decitabine lowered H3Y41ph and augmented H3K9me2 levels at the NFE2 locus in HEL cells, thereby increasing HP1α binding, which normalized NFE2 expression selectively in JAK2V617F-positive cell lines.

The cytoprotective role of DJ-1 and p45 NFE2 against human primary alveolar type II cell injury and emphysema.

Emphysema is characterized by irreversibly enlarged airspaces and destruction of alveolar walls. One of the factors contributing to this disease pathogenesis is an elevation in extracellular matrix (ECM) degradation in the lung. Alveolar type II (ATII) cells produce and secrete pulmonary surfactants and proliferate to restore the epithelium after damage. We isolated ATII cells from control non-smokers, smokers and patients with emphysema to determine the role of NFE2 (nuclear factor, erythroid-derived 2). NFE2 is a heterodimer composed of two subunits, a 45 kDa (p45 NFE2) and 18 kDa (p18 NFE2) polypeptides. Low expression of p45 NFE2 in patients with emphysema correlated with a high ECM degradation. Moreover, we found that NFE2 knockdown increased cell death induced by cigarette smoke extract. We also studied the cross talk between p45 NFE2 and DJ-1. DJ-1 protein is a redox-sensitive chaperone that protects cells from oxidative stress. We detected that cigarette smoke significantly increased p45 NFE2 levels in DJ-1 KO mice compared to wild-type mice. Our results indicate that p45 NFE2 expression is induced by exposure to cigarette smoke, has a cytoprotective activity against cell injury, and its downregulation in human primary ATII cells may contribute to emphysema pathogenesis.

Platelet-predominate gene expression and reticulated platelets in nonvalvular atrial fibrillation: Effect of pulmonary veins isolation.

INTRODUCTION: Reticulated platelet (RP) content is increased in nonvalvular atrial fibrillation (NVAF). The purpose of this study was to determine if platelet content, morphology, and RP proportion are modulated by platelet genes. METHODS AND RESULTS: Expression of six platelet-predominate genes impacting platelet formation and release, platelet count, and RP content was assessed in NVAF patients before and 3-4 months after pulmonary veins isolation (PVI) and compared to normal sinus rhythm (NSR) controls. RNA from isolated platelets was reverse-transcribed assayed against selected genes utilizing real-time qPCR, and expressed as mean cycle threshold (ΔCt) using beta-2-microglobulin as endogenous control. RP content was assessed by flow cytometry. A fourfold lower expression of CFL1 gene coding for nonmuscle cofilin (7.8 ± 0.9 vs. 5.7 ± 1.6, P < 0.001) and twofold lower expression of four other genes were associated with similar platelet counts but fourfold higher (28.7+7.0 vs. 6.7+5.4, P < 0.001) RP content (%) in 97 NVAF cases compared to 51 NSR controls. Three to 4 months after PVI, RP decreased by 28%, while CFL1 gene expression increased over twofold but TUBA4A gene expression decreased almost twofold; NFE2 and MYL6 gene expression remained unchanged. CONCLUSIONS: NVAF is associated with notable downregulation of genes directing platelet production and size but increased RP content. PVI impacts the expression of many of these genes, implying a direct relationship between atrial fibrillation and platelet biogenesis.

ITR­284 modulates cell differentiation in human chronic myelogenous leukemia K562 cells.

ITR­284 is a carboxamide analog that can inhibit proliferation in human promyelocytic leukemia HL-60 cells. To understand the effects and molecular mechanisms of ITR­284 in human erythromyeloblastoid leukemia, we treated K562 cells with different concentrations of ITR­284 (0, 2, 4, 6, 8 and 10 nM) and all-trans retinoic acid (ATRA) (0, 0.1, 0.5, 1, 5 and 10 µM) for 24 h. The IC50 of ITR­284 was ~10 nM in K562 cells treated for 24 h as determined by MTT assay. May-Grünwald-Giemsa staining and nitro blue tetrazolium (NBT) assays were used to determine cell morphology changes and differentiation after ITR­284 and ATRA treatment. In addition, mRNA expression levels of hematopoietic factors, including GATA­1, NF-E2 and GATA­2, were elevated, while expression levels of BCR­ABL were downregulated in K562 cells after 24 h of treatment with ITR­284 as determined by quantitative reverse transcription polymerase chain reaction. In addition, western blot analyses showed that FOXM1, GLI 1 and c-MYC protein levels were decreased by ITR­284. Taken together, our data show that ITR­284 induced K562 cell differentiation, which led to decreased tumorigenesis. Our findings suggest that ITR­284 could be a potential candidate for treating chronic myelogenous leukemia.

Antioxidant Effect of Barley Sprout Extract via Enhancement of Nuclear Factor-Erythroid 2 Related Factor 2 Activity and Glutathione Synthesis.

We previously showed that barley sprout extract (BSE) prevents chronic alcohol intake-induced liver injury in mice. BSE notably inhibited glutathione (GSH) depletion and increased inflammatory responses, revealing its mechanism of preventing alcohol-induced liver injury. In the present study we investigated whether the antioxidant effect of BSE involves enhancing nuclear factor-erythroid 2 related factor 2 (Nrf2) activity and GSH synthesis to inhibit alcohol-induced oxidative liver injury. Mice fed alcohol for four weeks exhibited significantly increased oxidative stress, evidenced by increased malondialdehyde (MDA) level and 4-hydroxynonenal (4-HNE) immunostaining in the liver, whereas treatment with BSE (100 mg/kg) prevented these effects. Similarly, exposure to BSE (0.1-1 mg/mL) significantly reduced oxidative cell death induced by t-butyl hydroperoxide (t-BHP, 300 µM) and stabilized the mitochondrial membrane potential (∆ψ). BSE dose-dependently increased the activity of Nrf2, a potential transcriptional regulator of antioxidant genes, in HepG2 cells. Therefore, increased expression of its target genes, heme oxygenase-1 (HO-1), NADPH quinone oxidoreductase 1 (NQO1), and glutamate-cysteine ligase catalytic subunit (GCLC) was observed. Since GCLC is involved in the rate-limiting step of GSH synthesis, BSE increased the GSH level and decreased both cysteine dioxygenase (CDO) expression and taurine level. Because cysteine is a substrate for both taurine and GSH synthesis, a decrease in CDO expression would further contribute to increased cysteine availability for GSH synthesis. In conclusion, BSE protected the liver cells from oxidative stress by activating Nrf2 and increasing GSH synthesis.

Changes in DNA methylation of erythroid-specific genes in K562 cells exposed to catechol in long term.

Catechol is one of phenolic metabolites of benzene that is a general occupational hazard and a ubiquitous environmental air pollutant. Catechol also occurs naturally in fruits, vegetables and cigarettes. Previous studies have revealed that 72h exposure to catechol improved hemin-induced erythroid differentiation of K562 cells accompanied with elevated methylation in erythroid specific genes. In present study, K562 cells were treated with 0, 10 or 20µM catechol for 1-4weeks, hemin-induced hemoglobin synthesis increased in a concentration- and time-dependent manner and the enhanced hemoglobin synthesis was relatively stable. The mRNA expression of α-, ß- and γ-globin genes, erythroid heme synthesis enzymes PBGD and ALAS2, transcription factor GATA-1 and NF-E2 showed a significant increase in K562 cells exposed to 20µM catechol for 3w, and catechol enhanced hemin-induced mRNA expression of these genes. Quantitative MassARRAY methylation analysis also confirmed that the exposure to catechol changed DNA methylation levels at several CpG sites in several erythroid-specific genes and their far upstream of regulatory elements. These results demonstrated that long-term exposure to low concentration of catechol enhanced the hemin-induced erythroid differentiation of K562 cells, in which DNA methylation played a role by up-regulating erythroid specific genes.