Courtney G. Woods

A systems biology perspective on Nrf2-mediated antioxidant response

Cells in vivo are constantly exposed to reactive oxygen species (ROS) generated endogenously and exogenously. To defend against the deleterious consequences of ROS, cells contain multiple antioxidant enzymes expressed in various cellular compartments to scavenge these toxic species. Under oxidative stresses, these antioxidant enzymes are upregulated to restore redox homeostasis. Such an adaptive response results from the activation of a redox-sensitive gene regulatory network mediated by nuclear factor E2-related factor 2. To more completely understand how the redox control system is designed by nature to meet homeostatic goals, we have examined the network from a systems perspective using engineering approaches. As with man-made control devices, the redox control system can be decomposed into distinct functional modules, including transducer, controller, actuator, and plant. Cells achieve specific performance objectives by utilizing nested feedback loops, feedforward control, and ultrasensitive signaling motifs, etc. Given that endogenously generated ROS are also used as signaling molecules, our analysis suggests a novel mode of action to explain oxidative stress-induced pathological conditions and diseases. Specifically, by adaptively upregulating antioxidant enzymes, oxidative stress may inadvertently attenuate ROS signals that mediate physiological processes, resulting in aberrations of cellular functions and adverse consequences. Lastly, by simultaneously considering the two competing cellular tasks-adaptive antioxidant defense and ROS signaling-we re-examine the premise that dietary antioxidant supplements is generally beneficial to human health. Our analysis highlights some possible adverse effects of these widely consumed antioxidants.

Low-Level Arsenic Impairs Glucose-Stimulated Insulin Secretion in Pancreatic Beta Cells: Involvement of Cellular Adaptive Response to Oxidative Stress

Background Chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with incidence of type 2 diabetes (T2D). A key driver in the pathogenesis of T2D is impairment of pancreatic β-cell function, with the hallmark of β-cell function being glucose-stimulated insulin secretion (GSIS). Reactive oxygen species (ROS) derived from glucose metabolism serve as one of the metabolic signals for GSIS. Nuclear factor-erythroid 2–related factor 2 (Nrf2) is a central transcription factor regulating cellular adaptive response to oxidative stress. Objectives We tested the hypothesis that activation of Nrf2 and induction of antioxidant enzymes in response to arsenic exposure impedes glucose-triggered ROS signaling and thus GSIS. Methods and results Exposure of INS-1(832/13) cells to low levels of arsenite led to decreased GSIS in a dose- and time-dependent fashion. Consistent with our hypothesis, a significantly enhanced Nrf2 activity, determined by its nuclear accumulation and induction of its target genes, was observed in arsenite-exposed cells. In keeping with the activation of Nrf2-mediated antioxidant response, intracellular glutathione and intracellular hydrogen peroxide–scavenging activity was dose dependently increased by arsenite exposure. Although the basal cellular peroxide level was significantly enhanced, the net percentage increase in glucose-stimulated intracellular peroxide production was markedly inhibited in arsenite-exposed cells. In contrast, insulin synthesis and the consensus GSIS pathway, including glucose transport and metabolism, were not significantly reduced by arsenite exposure. Conclusions Our studies suggest that low levels of arsenic provoke a cellular adaptive oxidative stress response that increases antioxidant levels, dampens ROS signaling involved in GSIS, and thus disturbs β-cell function.

Nuclear factor erythroid-derived factor 2-related factor 2 regulates transcription of CCAAT/enhancer-binding protein β during adipogenesis

Nuclear factor erythroid-derived factor 2-related factor 2 (Nrf2) is a cap-n-collar basic leucine zipper transcription factor that is involved in the cellular adaptive response to oxidative stress. Our previous study reported that targeted disruption of the Nrf2 gene in mice decreases adipose tissue mass and protects against obesity induced by a high-fat diet. Deficiency of Nrf2 in preadipocytes and mouse embryonic fibroblasts led to impaired adipogenesis. Consistent with these findings, the current study found that lack of Nrf2 in primary cultured mouse preadipocytes and 3T3-L1 cells hampered adipogenic differentiation induced by hormonal cocktails. Stable knockdown of Nrf2 in 3T3-L1 cells blocked the enhanced adipogenesis caused by deficiency of kelch-like ECH-associated protein 1 (Keap1), a Cul3-adapter protein that allows for Nrf2 to be ubiquinated and degraded by the 26S protesome complex. In addition, increased production of reactive oxygen species (ROS) and activation of Nrf2 occurred at the very early stage upon adipogenic hormonal challenge in 3T3-L1 cells, followed by an immediate induction of CCAAT/enhancer-binding protein β (C/EBPβ). Knockdown of Nrf2 led to reduced expression of C/EBPβ induced by adipogenic hormonal cocktails, chemical Nrf2 activators or Keap1 silencing. Cebpβ promoter-driven reporter assays and chromatin immunoprecipitation suggested that Nrf2 associates with a consensus antioxidant response element (ARE) binding site in the promoter of the Cebpβ gene during adipogenesis and upregulates its expression. These findings demonstrate a novel role of Nrf2 beyond xenobiotic detoxification and antioxidant response, and suggest that Nrf2 is one of the transcription factors that control the early events of adipogenesis by regulating expression of Cebpβ.

Adipose Deficiency of Nrf2 in ob/ob Mice Results in Severe Metabolic Syndrome

Nuclear factor E2–related factor 2 (Nrf2) is a transcription factor that functions as a master regulator of the cellular adaptive response to oxidative stress. Our previous studies showed that Nrf2 plays a critical role in adipogenesis by regulating expression of CCAAT/enhancer-binding protein β and peroxisome proliferator–activated receptor γ. To determine the role of Nrf2 in the development of obesity and associated metabolic disorders, the incidence of metabolic syndrome was assessed in whole-body or adipocyte-specific Nrf2-knockout mice on a leptin-deficient ob/ob background, a model with an extremely positive energy balance. On the ob/ob background, ablation of Nrf2, globally or specifically in adipocytes, led to reduced white adipose tissue (WAT) mass, but resulted in an even more severe metabolic syndrome with aggravated insulin resistance, hyperglycemia, and hypertriglyceridemia. Compared with wild-type mice, WAT of ob/ob mice expressed substantially higher levels of many genes related to antioxidant response, inflammation, adipogenesis, lipogenesis, glucose uptake, and lipid transport. Absence of Nrf2 in WAT resulted in reduced expression of most of these factors at mRNA or protein levels. Our findings support a novel role for Nrf2 in regulating adipose development and function, by which Nrf2 controls the capacity of WAT expansion and insulin sensitivity and maintains glucose and lipid homeostasis.

Dose-dependent transitions in Nrf2-mediated adaptive response and related stress responses to hypochlorous acid in mouse macrophages.

Hypochlorous acid (HOCl) is potentially an important source of cellular oxidative stress. Human HOCl exposure can occur from chlorine gas inhalation or from endogenous sources of HOCl, such as respiratory burst by phagocytes. Transcription factor Nrf2 is a key regulator of cellular redox status and serves as a primary source of defense against oxidative stress. We recently demonstrated that HOCl activates Nrf2-mediated antioxidant response in cultured mouse macrophages in a biphasic manner. In an effort to determine whether Nrf2 pathways overlap with other stress pathways, gene expression profiling was performed in RAW 264.7 macrophages exposed to HOCl using whole genome mouse microarrays. Benchmark dose (BMD) analysis on gene expression data revealed that Nrf2-mediated antioxidant response and protein ubiquitination were the most sensitive biological pathways that were activated in response to low concentrations of HOCl (<0.35 mM). Genes involved in chromatin architecture maintenance and DNA-dependent transcription were also sensitive to very low doses. Moderate concentrations of HOCl (0.35 to 1.4 mM) caused maximal activation of the Nrf2 pathway and innate immune response genes, such as IL-1beta, IL-6, IL-10 and chemokines. At even higher concentrations of HOCl (2.8 to 3.5 mM) there was a loss of Nrf2-target gene expression with increased expression of numerous heat shock and histone cluster genes, AP-1-family genes, cFos and Fra1 and DNA damage-inducible Gadd45 genes. These findings confirm an Nrf2-centric mechanism of action of HOCl in mouse macrophages and provide evidence of interactions between Nrf2, inflammatory, and other stress pathways.

Prolonged inorganic arsenite exposure suppresses insulin-stimulated AKT S473 phosphorylation and glucose uptake in 3T3-L1 adipocytes: Involvement of the adaptive antioxidant response

There is growing evidence that chronic exposure of humans to inorganic arsenic, a potent environmental oxidative stressor, is associated with the incidence of type 2 diabetes (T2D). One critical feature of T2D is insulin resistance in peripheral tissues, especially in mature adipocytes, the hallmark of which is decreased insulin-stimulated glucose uptake (ISGU). Despite the deleterious effects of reactive oxygen species (ROS), they have been recognized as a second messenger serving an intracellular signaling role for insulin action. Nuclear factor erythroid 2-related factor 2 (NRF2) is a central transcription factor regulating cellular adaptive response to oxidative stress. This study proposes that in response to arsenic exposure, the NRF2-mediated adaptive induction of endogenous antioxidant enzymes blunts insulin-stimulated ROS signaling and thus impairs ISGU. Exposure of differentiated 3T3-L1 cells to low-level (up to 2 μM) inorganic arsenite (iAs³(+)) led to decreased ISGU in a dose- and time-dependent manner. Concomitant to the impairment of ISGU, iAs³(+) exposure significantly attenuated insulin-stimulated intracellular ROS accumulation and AKT S473 phosphorylation, which could be attributed to the activation of NRF2 and induction of a battery of endogenous antioxidant enzymes. In addition, prolonged iAs³(+) exposure of 3T3-L1 adipocytes resulted in significant induction of inflammatory response genes and decreased expression of adipogenic genes and glucose transporter type 4 (GLUT4), suggesting chronic inflammation and reduction in GLUT4 expression may also be involved in arsenic-induced insulin resistance in adipocytes. Taken together our studies suggest that prolonged low-level iAs³(+) exposure activates the cellular adaptive oxidative stress response, which impairs insulin-stimulated ROS signaling that is involved in ISGU, and thus causes insulin resistance in adipocytes.

A map of the PPARα transcription regulatory network for primary human hepatocytes.

Nuclear receptor activation in liver leads to coordinated alteration of the expression of multiple gene products with attendant phenotypic changes of hepatocytes. Peroxisome proliferators including endogenous fatty acids, environmental chemicals, and drugs induce a multi-enzyme metabolic response that affects lipid and fatty acid processing. We studied the signaling network for the peroxisome proliferator-associated receptor alpha (PPARα) in primary human hepatocytes using the selective PPARα ligand, GW7647. We measured gene expression over multiple concentrations and times and conducted ChIP-seq studies at 2 and 24h to assess genomic binding of PPARα. Over all treatments there were 192 genes differentially expressed. Of these only 51% showed evidence of PPARα binding-either directly at PPARα response elements or via alternative mechanisms. Almost half of regulated genes had no PPARα binding. We then developed two novel bioinformatics methods to visualize the dose-dependent activation of both the transcription factor circuitry for PPARα and the downstream metabolic network in relation to functional annotation categories. Available databases identified several key transcription factors involved with the non-genomic targets after GW7647 treatment, including SP1, STAT1, ETS1, ERα, and HNF4α. The linkage from PPARα binding through gene expression likely requires intermediate protein kinases to activate these transcription factors. We found enrichment of functional annotation categories for organic acid metabolism and cell lipid metabolism among the differentially expressed genes. Lipid transport processes showed enrichment at the highest concentration of GW7647 (10 μM). While our strategy for mapping transcriptional networks is evolving, these approaches are necessary in moving from toxicogenomic methods that derive signatures of activity to methods that establish pathway structure, showing the coordination of the activated nuclear receptor with other signaling pathways.

Long Isoforms of NRF1 Contribute to Arsenic-Induced Antioxidant Response in Human Keratinocytes

Background Human exposure to inorganic arsenic (iAs), a potent oxidative stressor, causes various dermal disorders, including hyperkeratosis and skin cancer. Nuclear factor–erythroid 2–related factor 1 (NRF1, also called NFE2L1) plays a critical role in regulating the expression of many antioxidant response element (ARE)-dependent genes. Objectives We investigated the role of NRF1 in arsenic-induced antioxidant response and cytotoxicity in human keratinocytes. Results In cultured human keratinocyte HaCaT cells, inorganic arsenite (iAs3+) enhanced the protein accumulation of long isoforms (120–140 kDa) of NRF1 in a dose- and time-dependent fashion. These isoforms accumulated mainly in the nuclei of HaCaT cells. Selective deficiency of NRF1 by lentiviral short-hairpin RNAs in HaCaT cells [NRF1-knockdown (KD)] led to decreased expression of γ-glutamate cysteine ligase catalytic subunit (GCLC) and regulatory subunit (GCLM) and a reduced level of intracellular glutathione. In response to acute iAs3+ exposure, induction of some ARE-dependent genes, including NAD(P)H:quinone oxidoreductase 1 (NQO1), GCLC, and GCLM, was significantly attenuated in NRF1-KD cells. However, the iAs3-induced expression of heme oxygenase 1 (HMOX-1) was unaltered by silencing NRF1, suggesting that HMOX-1 is not regulated by NRF1. In addition, the lack of NRF1 in HaCaT cells did not disturb iAs3+-induced NRF2 accumulation but noticeably decreased Kelch-like ECH-associated protein 1 (KEAP1) levels under basal and iAs3+-exposed conditions, suggesting a potential interaction between NRF1 and KEAP1. Consistent with the critical role of NRF1 in the transcriptional regulation of some ARE-bearing genes, knockdown of NRF1 significantly increased iAs3+-induced cytotoxicity and apoptosis. Conclusions Here, we demonstrate for the first time that long isoforms of NRF1 contribute to arsenic-induced antioxidant response in human keratinocytes and protect the cells from acute arsenic cytotoxicity.

Genomic Profiling in Nuclear Receptor-Mediated Toxicity

Nuclear receptors (NRs) are attractive drug targets due to their role in regulation of a wide range of physiologic responses. In addition to providing therapeutic value, many pharmaceutical agents along with environmental chemicals are ligands for NRs and can cause adverse health effects that are directly related to activation of NRs. Identifying the molecular events that produce a toxic response may be confounded by the fact that there is a significant overlap in the biological processes that NRs regulate. Microarrays and other methods for gene expression profiling have served as useful, sensitive tools for discerning the mechanisms by which therapeutics and environmental chemicals invoke toxic effects. The capability to probe thousands of genes simultaneously has made genomics a prime technology for identifying drug targets, biomarkers of exposure/toxicity and key players in the mechanisms of disease. The complex intertwining networks regulated by NRs are hard to probe comprehensively without global approaches and genomics has become a key technology that facilitates our understanding of NR-dependent and -independent events. The future of drug discovery, design and optimization, and risk assessment of chemical toxicants that activate NRs will inevitably involve genomic profiling. This review will focus on genomics studies related to PPAR, CAR, PXR, RXR, LXR, FXR, and AHR.

Modeling Drug- and Chemical-Induced Hepatotoxicity with Systems Biology Approaches

We provide an overview of computational systems biology approaches as applied to the study of chemical- and drug-induced toxicity. The concept of “toxicity pathways” is described in the context of the 2007 US National Academies of Science report, “Toxicity testing in the 21st Century: A Vision and A Strategy.” Pathway mapping and modeling based on network biology concepts are a key component of the vision laid out in this report for a more biologically based analysis of dose-response behavior and the safety of chemicals and drugs. We focus on toxicity of the liver (hepatotoxicity) – a complex phenotypic response with contributions from a number of different cell types and biological processes. We describe three case studies of complementary multi-scale computational modeling approaches to understand perturbation of toxicity pathways in the human liver as a result of exposure to environmental contaminants and specific drugs. One approach involves development of a spatial, multicellular “virtual tissue” model of the liver lobule that combines molecular circuits in individual hepatocytes with cell–cell interactions and blood-mediated transport of toxicants through hepatic sinusoids, to enable quantitative, mechanistic prediction of hepatic dose-response for activation of the aryl hydrocarbon receptor toxicity pathway. Simultaneously, methods are being developing to extract quantitative maps of intracellular signaling and transcriptional regulatory networks perturbed by environmental contaminants, using a combination of gene expression and genome-wide protein-DNA interaction data. A predictive physiological model (DILIsym™) to understand drug-induced liver injury (DILI), the most common adverse event leading to termination of clinical development programs and regulatory actions on drugs, is also described. The model initially focuses on reactive metabolite-induced DILI in response to administration of acetaminophen, and spans multiple biological scales.