Coordinated regulation of Nrf2 and histone H3 serine 10 phosphorylation in arsenite-activated transcription of the human heme oxygenase-1 gene.

Expression of the antioxidant gene heme oxygenase-1 (HO-1) is primarily induced through NF-E2-related factor 2 (Nrf2)-mediated activation of the antioxidant response element (ARE). Gene transcription is coordinately regulated by transcription factor activity at enhancer elements and epigenetic alterations such as the posttranslational modification of histone proteins. However, the role of histone modifications in the Nrf2-ARE axis remains largely uncharacterized. The environmental contaminant arsenite is a potent inducer of both HO-1 expression and phosphorylation of histone H3 serine 10 (H3S10); therefore, we investigated the relationships between Nrf2 and H3S10 phosphorylation in arsenite-induced, ARE-dependent, transcriptional activation of the human HO-1 gene. Arsenite increased phosphorylation of H3S10 both globally and at the HO-1 promoter concomitantly with HO-1 transcription in human HaCaT keratinocytes. Conversely, arsenite-induced H3S10 phosphorylation and HO-1 expression were blocked by N-acetylcysteine (NAC), the c-Jun N-terminal kinase (JNK) inhibitor SP600125, and JNK knockdown (siJNK). Interestingly, ablation of arsenite-induced H3S10 phosphorylation by SP600125 or siJNK did not inhibit Nrf2 nuclear accumulation nor ARE binding, despite inhibiting HO-1 expression. In response to arsenite, binding of Nrf2 to the HO-1 ARE preceded phosphorylation of H3S10 at the HO-1 ARE. Furthermore, arsenite-mediated occupancy of phosphorylated H3S10 at the HO-1 ARE was decreased in Nrf2-deficient mouse embryonic fibroblasts. These results suggest the involvement of H3S10 phosphorylation in the Nrf2-ARE axis by proposing that Nrf2 may influence H3S10 phosphorylation at the HO-1 ARE and additional promoter regions. Our data highlights the complex interplay between Nrf2 and H3S10 phosphorylation in arsenite-activated HO-1 transcription.

Toxicological responses of environmental mixtures: Environmental metal mixtures display synergistic induction of metal-responsive and oxidative stress genes in placental cells.

Exposure to elevated levels of the toxic metals inorganic arsenic (iAs) and cadmium (Cd) represents a major global health problem. These metals often occur as mixtures in the environment, creating the potential for interactive or synergistic biological effects different from those observed in single exposure conditions. In the present study, environmental mixtures collected from two waste sites in China and comparable mixtures prepared in the laboratory were tested for toxicogenomic response in placental JEG-3 cells. These cells serve as a model for evaluating cellular responses to exposures during pregnancy. One of the mixtures was predominated by iAs and one by Cd. Six gene biomarkers were measured in order to evaluate the effects from the metal mixtures using dose and time-course experiments including: heme oxygenase 1 (HO-1) and metallothionein isoforms (MT1A, MT1F and MT1G) previously shown to be preferentially induced by exposure to either iAs or Cd, and metal transporter genes aquaporin-9 (AQP9) and ATPase, Cu(2+) transporting, beta polypeptide (ATP7B). There was a significant increase in the mRNA expression levels of ATP7B, HO-1, MT1A, MT1F, and MT1G in mixture-treated cells compared to the iAs or Cd only-treated cells. Notably, the genomic responses were observed at concentrations significantly lower than levels found at the environmental collection sites. These data demonstrate that metal mixtures increase the expression of gene biomarkers in placental JEG-3 cells in a synergistic manner. Taken together, the data suggest that toxic metals that co-occur may induce detrimental health effects that are currently underestimated when analyzed as single metals.

Identification of novel gene targets and putative regulators of arsenic-associated DNA methylation in human urothelial cells and bladder cancer.

There is strong epidemiologic evidence linking chronic exposure to inorganic arsenic (iAs) to myriad adverse health effects, including cancer of the bladder. We set out to identify DNA methylation patterns associated with arsenic and its metabolites in exfoliated urothelial cells (EUCs) that originate primarily from the urinary bladder, one of the targets of arsenic-induced carcinogenesis. Genome-wide, gene-specific promoter DNA methylation levels were assessed in EUCs from 46 residents of Chihuahua, Mexico, and the relationship was examined between promoter methylation profiles and the intracellular concentrations of total arsenic and arsenic species. A set of 49 differentially methylated genes was identified with increased promoter methylation associated with EUC tAs, iAs, and/or monomethylated As (MMAs) enriched for their roles in metabolic disease and cancer. Notably, no genes had differential methylation associated with EUC dimethylated As (DMAs), suggesting that DMAs may influence DNA methylation-mediated urothelial cell responses to a lesser extent than iAs or MMAs. Further analysis showed that 22 of the 49 arsenic-associated genes (45%) are also differentially methylated in bladder cancer tissue identified using The Cancer Genome Atlas repository. Both the arsenic- and cancer-associated genes are enriched for the binding sites of common transcription factors known to play roles in carcinogenesis, demonstrating a novel potential mechanistic link between iAs exposure and bladder cancer.

Transcriptional regulation of the human ferritin gene by coordinated regulation of Nrf2 and protein arginine methyltransferases PRMT1 and PRMT4.

Antioxidant genes such as ferritin are transcriptionally activated in oxidative stress via the antioxidant responsive element (ARE), to which nuclear factor-E2-related factor 2 (Nrf2) binds and activates transcription. Histone modification plays a cooperative and essential role in transcriptional regulation; however, its role in antioxidant gene transcription remains elusive. Arsenic exposure activated ferritin transcription via the ARE concomitant with increased methylation of histones H4Arg3 (H4R3) and H3Arg17 (H3R17). To test our hypothesis that histone H4R3 and H3R17 methylation regulates ferritin transcription, H4R3 and H3R17 protein arginine (R) methyltransferases 1 and 4 (PRMT1 and PRMT4) were investigated. Arsenic exposure of human HaCaT keratinocytes induced nuclear accumulation of PRMT1 and PRMT4, histone H4R3 and H3R17 methylation proximal to the ARE, but not to the non-ARE regions of ferritin genes. PRMT1 or PRMT4 knockdown did not block Nrf2 nuclear accumulation but inhibited Nrf2 binding to the AREs by ∼40% (P<0.05), thus diminishing ferritin transcription in HaCaT and human primary keratinocytes and fibroblasts, causing enhanced cellular susceptibility to arsenic toxicity as evidenced by 2-fold caspase 3 activation. Focused microarray further characterized several oxidative stress response genes are subject to PRMT1 or PRMT4 regulation. Collectively, PRMT1 and PRMT4 regulate the ARE and cellular antioxidant response to arsenic.

Role of AMP-activated protein kinase in ferritin H gene expression by resveratrol in human T cells.

Resveratrol, a natural polyphenol, increases cellular antioxidant capacity by inducing the expression of a battery of cytoprotective genes through an antioxidant responsive element (ARE). However, upstream signaling events initiated by resveratrol leading to the activation of an ARE enhancer, particularly in immune cells, have not been fully elucidated. In this study, ARE-dependent transcriptional activation of the ferritin heavy chain (ferritin H) gene by resveratrol was further investigated in Jurkat T cells and human peripheral blood mononuclear cells. We found that AMP-activated protein kinase (AMPK) plays a key role in the activation of nuclear factor E2-related factor (Nrf2) and subsequent ARE-dependent ferritin H gene transcription by resveratrol. A chromatin immunoprecipitation assay for Nrf2 after AMPKα knockdown with siRNA revealed that Nrf2 nuclear accumulation and subsequent binding to the ferritin H ARE induced by resveratrol were dependent on activation of AMPKα, but not PI3K/AKT. Furthermore, AMPKα knockdown blocked resveratrol-induced phosphorylation of glycogen synthase kinase 3ß (GSK3ß) at Ser9 as well as ARE-dependent transcriptional activation of the ferritin H and HO-1 genes, suggesting that AMPKα is an upstream kinase for GSK3ß phosphorylation and activation of the Nrf2-ARE pathway. Consistently, GSK3ß knockdown by siRNA enhanced resveratrol-mediated ferritin H mRNA induction, and the inhibition of AMPKα by compound C or siRNA weakened the protective effect of resveratrol against oxidative stress-induced cytotoxicity in CD3+ T cells. Collectively, these results suggest that AMPKα plays a significant role in ARE-dependent transcription of ferritin H genes by resveratrol and may influence the redox status in immune cells.

Role and regulation of ferritin H in rotenone-mediated mitochondrial oxidative stress.

Tight regulation of intracellular iron levels in response to mitochondrial dysfunction is an important mechanism that prevents oxidative stress, thereby limiting cellular damage. Here, we describe a cytoprotective response involving transcriptional activation of the ferritin H gene in response to the mitochondrial complex I inhibitor and neurotoxic compound rotenone. Rotenone exposure increased ferritin H mRNA and protein synthesis in NIH3T3 fibroblasts and SH-SY5Y neuroblastoma cells. Transient transfection of a ferritin H promoter-luciferase reporter into NIH3T3 cells showed that ferritin H was transcriptionally activated by rotenone through an antioxidant-responsive element (ARE). Chromatin immunoprecipitation assays showed that rotenone treatment enhanced binding of Nrf2 and JunD transcription factors to the ARE. In addition, rotenone induced production of reactive oxygen species (ROS), and pretreatment with N-acetylcysteine abrogated ferritin H mRNA induction by rotenone, suggesting that this response is oxidative stress-mediated. Furthermore, reduced ferritin H expression by siRNA sensitized cells to rotenone-induced apoptosis with increased ROS production and annexin V-positive cells. Taken together, these results suggest that ferritin H transcription is activated by rotenone via an oxidative stress-mediated pathway leading to ARE activation and may be critically important to protect cells from mitochondrial dysfunction and oxidative stress.

Motor-like DNA motion due to an ATP-hydrolyzing protein under nanoconfinement.

We report that long double-stranded DNA confined to quasi-1D nanochannels undergoes superdiffusive motion under the action of the enzyme T4 DNA ligase in the presence of necessary co-factors. Inside the confined environment of the nanochannel, double-stranded DNA molecules stretch out due to self-avoiding interactions. In absence of a catalytically active enzyme, we see classical diffusion of the center of mass. However, cooperative interactions of proteins with the DNA can lead to directed motion of DNA molecules inside the nanochannel. Here we show directed motion in this configuration for three different proteins (T4 DNA ligase, MutS, E. coli DNA ligase) in the presence of their energetic co-factors (ATP, NAD+).

Epigenetics and Preeclampsia: Defining Functional Epimutations in the Preeclamptic Placenta Related to the TGF-ß Pathway.

Preeclampsia is a potentially fatal pregnancy disorder affecting millions of women around the globe. Dysregulation in gene and protein expression within key biological pathways controlling angiogenesis has been implicated in the development of preeclampsia. Altered CpG methylation, a type of epimutation, may underlie this pathway dysregulation. In the present study, placental tissue from preeclamptic cases and normotensive controls was analyzed for genome-wide differential CpG methylation and concomitant changes in gene expression. A set of 123 genes, representing 19.9% of all genes with altered CpG methylation, was associated with functional changes in transcript levels. Underscoring the complex relationships between CpG methylation and gene expression, here hypermethylation was never associated with gene silencing, nor was hypomethylation always associated with gene activation. Moreover, the genomic region of the CpG mark was important in predicting the relationship between CpG methylation and gene expression. The 123 genes were enriched for their involvement in the transforming growth factor beta (TGF-ß) signaling pathway, a known regulator of placental trophoblast invasion and migration. This is the first study to identify CpG hypomethylation as an activator of TGF-ß-associated gene expression in the preeclamptic placenta. The results suggest functional epimutations are associated with preeclampsia disease status and the identified genes may represent novel biomarkers of disease.

Incorporating epigenetic data into the risk assessment process for the toxic metals arsenic, cadmium, chromium, lead, and mercury: strategies and challenges.

Exposure to toxic metals poses a serious human health hazard based on ubiquitous environmental presence, the extent of exposure, and the toxicity and disease states associated with exposure. This global health issue warrants accurate and reliable models derived from the risk assessment process to predict disease risk in populations. There has been considerable interest recently in the impact of environmental toxicants such as toxic metals on the epigenome. Epigenetic modifications are alterations to an individual's genome without a change in the DNA sequence, and include, but are not limited to, three commonly studied alterations: DNA methylation, histone modification, and non-coding RNA expression. Given the role of epigenetic alterations in regulating gene and thus protein expression, there is the potential for the integration of toxic metal-induced epigenetic alterations as informative factors in the risk assessment process. In the present review, epigenetic alterations induced by five high priority toxic metals/metalloids are prioritized for analysis and their possible inclusion into the risk assessment process is discussed.

Reactive oxygen species (ROS) homeostasis and redox regulation in cellular signaling.

Reactive oxygen species (ROS) are generated during mitochondrial oxidative metabolism as well as in cellular response to xenobiotics, cytokines, and bacterial invasion. Oxidative stress refers to the imbalance due to excess ROS or oxidants over the capability of the cell to mount an effective antioxidant response. Oxidative stress results in macromolecular damage and is implicated in various disease states such as atherosclerosis, diabetes, cancer, neurodegeneration, and aging. Paradoxically, accumulating evidence indicates that ROS also serve as critical signaling molecules in cell proliferation and survival. While there is a large body of research demonstrating the general effect of oxidative stress on signaling pathways, less is known about the initial and direct regulation of signaling molecules by ROS, or what we term the "oxidative interface." Cellular ROS sensing and metabolism are tightly regulated by a variety of proteins involved in the redox (reduction/oxidation) mechanism. This review focuses on the molecular mechanisms through which ROS directly interact with critical signaling molecules to initiate signaling in a broad variety of cellular processes, such as proliferation and survival (MAP kinases, PI3 kinase, PTEN, and protein tyrosine phosphatases), ROS homeostasis and antioxidant gene regulation (thioredoxin, peroxiredoxin, Ref-1, and Nrf-2), mitochondrial oxidative stress, apoptosis, and aging (p66Shc), iron homeostasis through iron-sulfur cluster proteins (IRE-IRP), and ATM-regulated DNA damage response.