Nuclear factor erythroid-derived 2 (NF-E2), a heterodimer composed of p45 and p18, is a transcriptional activator in hematopoietic progenitors. The transcriptional activity of NF-E2 is not only upregulated by SUMOylation but also stimulated by the cAMP-dependent protein kinase A (PKA). However, the relationship between SUMOylation and phosphorylation in the activation of NF-E2 is unclear. In the present studies, we have demonstrated that PKA enhances NF-E2 SUMOylation in an in vitro system using purified proteins, suggesting a possible mechanism for PKA-dependent activation of the NF-E2 transcription factor through SUMOylation.

The molecular etiology of myeloproliferative neoplasms (MPNs) remains incompletely understood, despite recent advances incurred through the discovery of several different mutations in MPN patients. We have recently described overexpression of the transcription factor NF-E2 in MPN patients and shown that elevated NF-E2 levels in vivo cause an MPN phenotype and predispose to leukemic transformation in transgenic mice. We report the presence of acquired insertion and deletion mutations in the NF-E2 gene in MPN patients. These result in truncated NF-E2 proteins that enhance wild-type (WT) NF-E2 function and cause erythrocytosis and thrombocytosis in a murine model. NF-E2 mutant cells acquire a proliferative advantage, witnessed by clonal dominance over WT NF-E2 cells in MPN patients. Our data underscore the role of increased NF-E2 activity in the pathophysiology of MPNs.

Platelet integrin alphaIIbbeta3 responds to intracellular signals by binding fibrinogen and triggering cytoskeletal reorganization, but the mechanisms of alphaIIbbeta3 signaling remain poorly understood. To better understand this process, we established conditions to study alphaIIbbeta3 signaling in primary murine megakaryocytes. Unlike platelets, these platelet precursors are amenable to genetic manipulation. Cytokine-stimulated bone marrow cultures produced three arbitrary populations of alphaIIbbeta3-expressing cells with increasing size and DNA ploidy: small progenitors, intermediate-size young megakaryocytes, and large mature megakaryocytes. A majority of the large megakaryocytes bound fibrinogen in response to agonists, while almost none of the smaller cells did. Fibrinogen binding to large megakaryocytes was inhibited by Sindbis virus-mediated expression of isolated beta3 integrin cytoplasmic tails. Strikingly, large megakaryocytes from mice deficient in the transcription factor NF-E2 failed to bind fibrinogen in response to agonists, despite normal surface expression of alphaIIbbeta3. Furthermore, while megakaryocytes from wild-type mice spread on immobilized fibrinogen and exhibited filopodia, lamellipodia and Rho-dependent focal adhesions and stress fibers, NF-E2-deficient megakaryocytes adhered poorly. These studies establish that agonist-induced activation of alphaIIbbeta3 is controlled by NF-E2-regulated signaling pathways that mature late in megakaryocyte development and converge at the beta3 cytoplasmic tail. Megakaryocytes provide a physiologically relevant and tractable system for analysis of bidirectional alphaIIbbeta3 signaling.

Radiation-induced skin injury (RISI) commonly occur in cancer patients who received radiotherapy and is one of the first clinical symptoms after suffering from nuclear exposure. Oxidative damage is the major causes of RISI. Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered as a key mediator of the cellular antioxidant response. However, whether Nrf2 can alleviate RISI after high-dose irradiation remains unknown. In this study, we demonstrated that Nrf2-deficient (Nrf2 -/-) mice were susceptible to high-dose irradiation and adenovirus-mediated overexpression of Nrf2 (ad-Nrf2) protected against radiation in skin cells. Overexpression of Nrf2 attenuated the severity of skin injury after high-dose electron beam irradiation. To uncover the mechanisms of Nrf2 involved in RISI, mRNA sequencing technology was performed to analyze the mRNA expression profiles of Ad-Nrf2 skin cells following radiation. The results revealed that a total of 127 genes were significantly changed, 55 genes were upregulated, and 72 genes were downregulated after Nrf2 overexpression. GSEA showed that Nrf2 was associated with positive regulation of genes involved in the reactive oxygen species pathway after radiation. Taken together, this study illustrated the role of Nrf2 in RISI and provided potentially strategies for ameliorating RISI.

Dendritic cells (DCs) are critical mediators of immunity and immune tolerance by orchestrating multiple aspects of T cell activation and function. Immature DCs (iDCs) expressing low levels of co-stimulatory receptors are highly efficient at antigen capture but are poor activators of T cells. Maturation of DCs is associated with increased expression of co-stimulatory molecules. Co-stimulatory receptor gene expression is regulated by intracellular redox, NF-κB, and MAPK pathways and by histone deacetylase (HDAC) activity. The transcription factor, Nrf2, is important for maintaining intracellular glutathione (GSH) levels and redox homeostasis and has been implicated in modulating DC co-stimulatory receptor expression. It is unclear whether Nrf2 mediates this effect by GSH-dependent mechanisms and whether it influences DC signaling pathways. Using bone marrow-derived iDCs from Nrf2(+/+) and Nrf2(-/-) mice, we demonstrate that Nrf2(-/-) iDCs have lower basal GSH levels, enhanced co-stimulatory receptor expression, impaired phagocytic functions, and increased antigen-specific CD8 T cell stimulation capacity. Interestingly, lowering GSH levels in Nrf2(+/+) iDCs did not recapitulate the Nrf2(-/-) iDC phenotype. Loss of Nrf2 resulted in elevated basal levels of reactive oxygen species but did not affect basal NF-κB activity or p38 MAPK phosphorylation. Using pharmacological inhibitors, we demonstrate that enhanced co-stimulatory receptor phenotype of Nrf2(-/-) iDC does not require ERK activity but is dependent on HDAC activity, indicating a potential interaction between Nrf2 function and HDAC. These results suggest that Nrf2 activity is required to counter rises in intracellular reactive oxygen species and to regulate pathways that control DC co-stimulatory receptor expression.

Free radicals generated by oxidative stress cause damage that can contribute to numerous chronic diseases. Mammalian cells respond to this damage by increased transcription of cytoprotective phase II genes, which are regulated by NRF2. Previously, it has been shown that NRF2 protein levels increase after oxidative stress because its negative regulator, KEAP1, loses its ability to bind NRF2 and cause its proteasome-mediated degradation during oxidative stress. Here, we show that CRIF1, a protein previously known as cell cycle regulator and transcription cofactor, is also able to negatively regulate NRF2 protein stability. However, in contrast to KEAP1, which regulates NRF2 stability only under normal reducing conditions, CRIF1 regulates NRF2 stability and its target gene expression under both reducing and oxidative stress conditions. Thus, CRIF1-NRF2 interactions and their consequences are redox-independent. In addition, we found that CRIF1, unlike KEAP1 (which only interacts with N-terminal region of NRF2), physically interacts with both N- and C-terminal regions of NRF2 and promotes NRF2 ubiquitination and subsequent proteasome-mediated NRF2 protein degradation.

It has been reported that activation of NF-E2 p45-related factor-2 (NRF2), a transcription factor, induces a variety of antioxidant enzymes, and plays an important role in preventing carcinogenesis. AHCC is a standardized extract of cultured Lentinula edodes mycelia and it has been demonstrated to improve cancer. However, the effects of AHCC on NRF2 have not been examined, and the effects on intestinal adenoma development are not yet fully understood. We first investigated the effects of AHCC (1-5 mg/ml) on NRF2 activity in human colon cancer cell lines by a luciferase reporter gene assay, and found NRF2 transcriptional activities were increased ~12.6-fold. In addition, AHCC dose-dependently increased HO-1 and NQO-1 mRNA levels, and decreased interleukine-6 mRNA levels. Next, we administered 1,000 ppm AHCC for 8 weeks in the diet of Apc mutant Min mice, and found that AHCC significantly reduced the total number of intestinal polyps to 57.7% and to 67.6% of the control value in male and female Min mice, respectively, with suppression of interleukine-6 in the polyp part. These data suggest that AHCC possesses an ability to suppress cellular oxidative stress through activation of NRF2, thereby lowering intestinal polyp development in Min mice.

Cereblon is a protein encoded by the CRBN gene, which has been associated with human autosomal recessive nonsyndromic mental retardation. However, little is known about the regulation of CRBN expression. Following exposure of mouse neuroblastoma N2A cells to hypoxia/reoxygenation (H/R), mRNA and protein expression of CRBN were increased. To better understand how CRBN expression is regulated, the promoter region of the mouse CRBN gene was characterized functionally. Deletion mutations and site-directed mutagenesis led to the identification of a functional NF-E2-related factor 2 (Nrf2)-binding site. Electrophoretic mobility shift analysis indicated that Nrf2 binds to a putative binding site in the CRBN promoter. Nrf2 overexpression and tert-butylhydroquinone treatment enhanced CRBN protein expression. These results imply that Nrf2 stimulates CRBN gene transcription under H/R conditions in neuronal cells.

Nonalcoholic steatohepatitis (NASH) is associated with oxidative stress. We surmised that pharmacologic activation of NF-E2 p45-related factor 2 (Nrf2) using the acetylenic tricyclic bis(cyano enone) TBE-31 would suppress NASH because Nrf2 is a transcriptional master regulator of intracellular redox homeostasis.

Oxidative stress has been implicated in the pathogenesis of Friedreich's Ataxia (FRDA), a neurodegenerative disease caused by the decreased expression of frataxin, a mitochondrial protein responsible of iron homeostasis. Under conditions of oxidative stress, the activation of the transcription factor NF-E2-related factor (Nrf2) triggers the antioxidant cellular response by inducing antioxidant response element (ARE) driven genes. Increasing evidence supports a role for the Nrf2-ARE pathway in neurodegenerative diseases. In this study, we analyzed the expression and the distribution of Nrf2 in silenced neurons for frataxin gene. Decreased Nrf2 mRNA content and a defective activation after treatment with pro-oxidants have been evidenced in frataxin-silenced neurons by RT-PCR and confocal microscopy. The loss of Nrf2 in FRDA may greatly enhance the cellular susceptibility to oxidative stress and make FRDA neurons more vulnerable to injury. Our findings may help to focus on this promising target, especially in its emerging role in the neuroprotective response.

Heat shock increases skin temperature during sun exposure and some evidence indicates that it may be involved in skin aging. The antioxidant response mediated by the transcription factor NF-E2-related factor 2 (Nrf2) is a critically important cellular defense mechanism that serves to limit skin aging. We investigated the effects of heat shock on collagenase expression when the antioxidant defense system was downregulated by knockdown of Nrf2. GSH and collagenases were analyzed, and the expression of inducible Nrf2, HO-1, and NQO1 was measured. HS68 cells were transfected with small interfering RNA against Nrf2. Heat shock induced the downregulation of Nrf2 in both the cytosol and nucleus and reduced the expression of HO-1, GSH, and NQO1. In addition, heat-exposed Nrf2-knockdown cells showed significantly increased levels of collagenase protein and decreased levels of procollagen. Our data suggest that Nrf2 plays an important role in protection against heat shock-induced collagen breakdown in skin.

Sulforaphane is an activator of transcription factor NF-E2-related factor-2 (nrf2) that regulates gene expression through the promoter antioxidant response element (ARE). Nrf2 regulates the transcription of a battery of protective and metabolic enzymes. The aim of this study was to assess whether activation of nrf2 by sulforaphane in human microvascular endothelial cells prevents metabolic dysfunction in hyperglycemia.

Selenium has been reported to induce the expression of some cytoprotective enzymes, which may account for its chemoprotective and chemopreventive effects. However, it remains largely unresolved whether these effects are exerted by selenium itself or mediated by its metabolite(s). In the present study, methylseleninic acid (MSeA), a monomethylated selenium, induced the expression of NAD(P)H:quinone oxidoreductase-1 (NQO-1) in human Chang liver cells. Expression of NQO-1 and other antioxidant/stress response genes is primarily regulated by the transcription factor NF-E2-related factor2 (Nrf2). Exposure of human Chang liver cells to MSeA (3 μM) increased nuclear translocation of Nrf2 and binding to antioxidant response elements. Silencing Nrf2 markedly reduced the MSeA-induced NQO-1 expression. In comparison with embryonic fibroblasts from Nrf2 wild-type mice, those from Nrf2 knockout mice failed to induce NQO-1 expression when treated with MSeA. Moreover, MSeA treatment enhanced ubiquitination of Keap1, but repressed Nrf2 ubiquitination. Pretreatment of cells with dithiothreitol abrogated the MSeA-induced NQO-1 expression, suggesting that MSeA causes Keap1 thiol modification. MSeA-induced NQO-1 upregulation was attenuated in cells harbouring the mutant Keap1 in which the cysteine 151 residue was replaced by serine. Oral administration of MSeA (1 mg/kg) by gavage to mice induced hepatic NQO-1 expression. Similar to MSeA, methylselenol generated from selenomethionine by methioninase activity induced NQO-1 expression. In conclusion, MSeA, the immediate precursor of methylselenol, upregulates the expression of NQO-1 via the Keap1-Nrf2 signaling. The above findings suggest that biological activities of selenium are dependent on the nature of the metabolites as well as the type of ingested selenium formulations.

Statin has been known not only as their cholesterol-lowering action but also on their pleiotropic effects including anti-inflammatory and anti-oxidant as well as anti-cancer effect. Nrf2 (NF-E2-related factor 2) is a transcription factor to activate cellular antioxidant response to oxidative stress. There are little known whether statins affect activation of Nrf2 and Nrf2 signaling pathway in colon cancer cells. We investigated whether simvastatin stimulates the expression of Nrf2 and nuclear translocation of Nrf2 and which signal pathway is involved in the expression of Nrf2 and antioxidant enzymes. We investigated the effect of simvastatin on the expression of Nrf2 and nuclear translocation of Nrf2 in two human colon cancer cell lines, HT-29 and HCT 116 through cell proliferation assay, Western blotting and immunocytochemical analysis. We evaluated which signal pathway such as ERK or PI3K pathway affect Nrf2 activation and whether simvastatin induces antioxidant enzymes (heme oxygenase-1 (HO-1), NAD(P)H: quinine oxidoreductase 1 (NQO1), γ-glutamate-cysteine ligase catalytic subunit (GCLC)). We demonstrated simvastatin-induced dose-dependent up-regulation of Nrf2 expression and stimulated Nrf2 nuclear translocation in colon cancer cells. We also demonstrated that simvastatin-induced anti-oxidant enzymes (HO-1, NQO1, and GCLC) in HT-29 and HCT 116 cells. PI3K/Akt inhibitor (LY294002) and ERK inhibitor (PD98059) suppressed simvastatin-induced Nrf2 and HO-1 expression in both HT-29 and HCT 116 cells. This study shows that simvastatin induces the activation and nuclear translocation of Nrf2 and the expression of various anti-oxidant enzymes via ERK and PI3K/Akt pathway in colon cancer cells.

Placental insufficiency jeopardizes prenatal development, potentially leading to intrauterine growth restriction (IUGR) and stillbirth. Surviving fetuses are at an increased risk for chronic diseases later in life. IUGR is closely linked with altered trophoblast and placental differentiation. However, due to a paucity of mechanistic insights, suitable biomarkers and specific therapies for IUGR are lacking. The transcription factor p45 NF-E2 (nuclear factor erythroid derived 2) has been recently found to regulate trophoblast differentiation in mice. The absence of p45 NF-E2 in trophoblast cells causes IUGR and placental insufficiency in mice, but mechanistic insights are incomplete and the relevance of p45 NF-E2 for human syncytiotrophoblast differentiation remains unknown. Here we show that p45 NF-E2 negatively regulates human syncytiotrophoblast differentiation and is associated with IUGR in humans. Expression of p45 NF-E2 is reduced in human placentae complicated with IUGR compared with healthy controls. Reduced p45 NF-E2 expression is associated with increased syncytiotrophoblast differentiation, enhanced glial cells missing-1 (GCM1) acetylation and GCM1 desumoylation in IUGR placentae. Induction of syncytiotrophoblast differentiation in BeWo and primary villous trophoblast cells with 8-bromo-adenosine 3',5'-cyclic monophosphate (8-Br-cAMP) reduces p45 NF-E2 expression. Of note, p45 NF-E2 knockdown is sufficient to increase syncytiotrophoblast differentiation and GCM1 expression. Loss of p45 NF-E2 using either approach resulted in CBP-mediated GCM1 acetylation and SENP-mediated GCM1 desumoylation, demonstrating that p45 NF-E2 regulates post-translational modifications of GCM1. Functionally, reduced p45 NF-E2 expression is associated with increased cell death and caspase-3 activation in vitro and in placental tissues samples. Overexpression of p45 NF-E2 is sufficient to repress GCM1 expression, acetylation and desumoylation, even in 8-Br-cAMP exposed BeWo cells. These results suggest that p45 NF-E2 negatively regulates differentiation and apoptosis activation of human syncytiotrophoblast by modulating GCM1 acetylation and sumoylation. These studies identify a new pathomechanism related to IUGR in humans and thus provide new impetus for future studies aiming to identify new biomarkers and/or therapies of IUGR.

MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate the expression of target messenger RNA (mRNA) complementary to the 3' untranslated region (UTR) at the post-transcriptional level. Hsa-miR-422a, which is commonly known as miRNA derived from transposable element (MDTE), was derived from short interspersed nuclear element (SINE). Through expression analysis, hsa-miR-422a was found to be highly expressed in both the small intestine and liver of crab-eating monkey. AT-Rich Interaction Domain 5 B (ARID5B) was selected as the target gene of hsa-miR-422a, which has two binding sites in both the exon and 3'UTR of ARID5B. To identify the interaction between hsa-miR-422a and ARID5B, a dual luciferase assay was conducted in HepG2 cell line. The luciferase activity of cells treated with the hsa-miR-422a mimic was upregulated and inversely downregulated when both the hsa-miR-422a mimic and inhibitor were administered. Nuclear factor erythroid-2 (NF-E2) was selected as the core transcription factor (TF) via feed forward loop analysis. The luciferase expression was downregulated when both the hsa-miR-422a mimic and siRNA of NF-E2 were treated, compared to the treatment of the hsa-miR-422a mimic alone. The present study suggests that hsa-miR-422a derived from SINE could bind to the exon region as well as the 3'UTR of ARID5B. Additionally, hsa-miR-422a was found to share binding sites in ARID5Bwith several TFs, including NF-E2. The hsa-miR-422a might thus interact with TF to regulate the expression of ARID5B, as demonstrated experimentally. Altogether, hsa-miR-422a acts as a super enhancer miRNA of ARID5Bby collaborating with TF and NF-E2.

Intestinal ischemia/reperfusion (IIR) is a common pathological event associated with intestinal injury and apoptosis with high mortality. Nuclear factor (NF)-E2-related factor-2 (Nrf2) is a key transcription factor that interacts with NF-κB and has a vital anti-inflammatory effect. However, whether Nrf2 has a role in IIR-induced apoptosis and the possible underlining mechanisms, such as modulation of the inflammation regulation pathway, have remained to be fully elucidated. In the present study, IIR was identified to cause significant intestinal injury and apoptosis, with high expression levels of inflammatory cytokines, as well as the apoptotic proteins B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) and caspase-3, while simultaneously decreasing the protein levels of Bcl-2. The effect was more pronounced after pretreatment of the animals with all-trans retinoic acid or brusatol, potent inhibitors of Nrf2. t-Butylhydroquinone, an Nrf2 activator, significantly attenuated IIR-induced intestinal injury and apoptosis, with inhibition of the overexpression of the inflammatory cytokines, Bax and caspase-3 protein and partial restoration of Bcl-2 protein expression. Taken together, these results indicated that increased Nrf2 expression reduced IIR-induced intestinal apoptosis and that the protective function of Nrf2 may be based on its anti-inflammatory effects through the inhibition of the NF-κB pathway.

The transcription factor NF-E2 Related Factor-2 (NRF2) is an important drug target. Activation of NRF2 has chemopreventive effects in cancer and exerts beneficial effects in a number of diseases, including neurodegenerative diseases, inflammatory diseases, hepatosteatosis, obesity and insulin resistance. Hence, there have been great efforts to discover and characterize novel NRF2 activators. One reported NRF2 activator is the labdane diterpenoid andrographolide. In this study, we identified the mechanism through which andrographolide activates NRF2. We showed that andrographolide inhibits the function of KEAP1, a protein that together with CUL3 and RBX1 forms an E3 ubiquitin ligase that polyubiquitinates NRF2. Andrographolide partially inhibits the interaction of KEAP1 with CUL3 in a manner dependent on Cys151 in KEAP1. This suggests that andrographolide forms Michael acceptor dependent adducts with Cys151 in KEAP1 in vivo, leading to inhibition of NRF2 ubiquitination and consequently accumulation of the transcription factor. Interestingly, we also showed that at higher concentrations andrographolide increases NRF2 protein expression in a Cys151 independent, but likely KEAP1 dependent manner, possibly through modification of other Cys residues in KEAP1. In this study we also screened secondary metabolites produced by endophytes isolated from non-flowering plants for NRF2-inducing properties. One of the extracts, ORX 41, increased both NRF2 protein expression and transcriptional activity markedly. These results suggest that endophytes isolated from non-flowering or other plants may be a good source of novel NRF2 inducing compounds.

The CNC-basic leucine zipper (CNC-bZIP) family is a subfamily of bZIP proteins identified from independent searches for factors that bind the AP-1-like cis-elements in the beta-globin locus control region. Three members, p45-Nf-e2, Nrf-1 and Nrf-2 have been identified in mammals. Expression of p45-Nf-e2 is largely restricted to hematopoietic cells while Nrf-1 and Nrf-2 are expressed in a wide range of tissues. To determine the function of Nrf-1, targeted disruption of the Nrf-1 gene was carried out. Homozygous Nrf-1 mutant mice are anemic due to a non-cell autonomous defect in definitive erythropoiesis and die in utero.

Inflammation plays a central role in the terminal processes of human labour and delivery, including the rupture of fetal membranes. Recent studies show a role for the transcription factor Nrf2 (NF-E2-related factor 2) in regulating inflammation. The aims of this study were to determine the effect of human spontaneous term and preterm labour on Nrf2 expression in fetal membranes; and Nrf2 siRNA knockdown on pro-inflammatory cytokines.