Jonathan M. Maher

Oxidative and electrophilic stress induces multidrug resistance–associated protein transporters via the nuclear factor‐E2–related factor‐2 transcriptional pathway

Multidrug resistance–associated proteins (Mrps) are adenosine triphosphate–dependent transporters that efflux chemicals out of cells. In the liver, Mrp2 transports bilirubin‐glucuronide, glutathione (GSH), and drug conjugates into bile, whereas Mrp3 and Mrp4 efflux these entities into blood. The purpose of this study was to determine whether oxidative conditions (that is, the disruption of hepatic GSH synthesis) or the administration of nuclear factor‐E2–related factor‐2 (Nrf2) activators (oltipraz and butylated hydroxyanisole) can induce hepatic Mrp transporters and whether that induction is through the Nrf2 transcriptional pathway. Livers from hepatocyte‐specific glutamate‐cysteine ligase catalytic subunit–null mice had increased nuclear Nrf2 levels, marked gene and protein induction of the Nrf2 target gene NAD(P)H:quinone oxidoreductase 1, as well as Mrp2, Mrp3, and Mrp4 expression. The treatment of wild‐type and Nrf2‐null mice with oltipraz and butylated hydroxyanisole demonstrated that the induction of Mrp2, Mrp3, and Mrp4 is Nrf2‐dependent. In Hepa1c1c7 cells treated with the Nrf2 activator tert‐butyl hydroquinone, chromatin immunoprecipitation with Nrf2 antibodies revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of mouse Mrp2 [−185 base pairs (bp)], Mrp3 (−9919 bp), and Mrp4 (−3767 bp). Conclusion: The activation of the Nrf2 regulatory pathway stimulates the coordinated induction of hepatic Mrps. (HEPATOLOGY 2007.)

The rise of antioxidant signaling-The evolution and hormetic actions of Nrf2

Organisms have evolved sophisticated and redundant mechanisms to manage oxidative and electrophilic challenges that arise from internal metabolism or xenobiotic challenge for survival. NF-E2-related factor 2 (Nrf2) is a transcription factor that has evolved over millennia from primitive origins, with homologues traceable back to invertebrate Caenorhabditis and Drosophila species. The ancestry of Nrf2 clearly has deep-seated roots in hematopoiesis, yet has diversified into a transcription factor that can mediate a multitude of antioxidant signaling and detoxification genes. In higher organisms, a more sophisticated means of tightly regulating Nrf2 activity was introduced via the cysteine-rich kelch-like ECH-associated protein 1 (Keap1), thus suggesting a need to modulate Nrf2 activity. This is evidenced in Keap1(-/-) mice, which succumb to juvenile mortality due to hyperkeratosis of the gastrointestinal tract. Although Nrf2 activation protects against acute toxicity and prevents or attenuates several disease states, constitutive activation in some tumors leads to poor clinical outcomes, suggesting Nrf2 has evolved in response to a multitude of selective pressures. The purpose of this review is to examine the origins of Nrf2, while highlighting the versatility and protective abilities elicited upon activation. Various model systems in which Nrf2 is normally beneficial but in which exaggerated pharmacology exacerbates a physiological or pathological condition will be addressed. Although Darwinian principles have selected Nrf2 activity for maximal beneficial effect based on environmental and oxidative challenge, both sub- or super-physiological effects have been noted to be detrimental. The functions of Nrf2 thus suggest a hormetic factor that has evolved empirically over time.

BRG1 Interacts with Nrf2 To Selectively Mediate HO-1 Induction in Response to Oxidative Stress

ABSTRACT NF-E2-related factor 2 (Nrf2) regulates antioxidant-responsive element-mediated induction of cytoprotective genes in response to oxidative stress. The purpose of this study was to determine the role of BRG1, a catalytic subunit of SWI2/SNF2-like chromatin-remodeling complexes, in Nrf2-mediated gene expression. Small interfering RNA knockdown of BRG1 in SW480 cells selectively decreased inducible expression of the heme oxygenase 1 (HO-1) gene after diethylmaleate treatment but did not affect other Nrf2 target genes, such as the gene encoding NADPH:quinone oxidoreductase 1 (NQO1). Chromatin immunoprecipitation analysis revealed that Nrf2 recruits BRG1 to both HO-1 and NQO1 regulatory regions. However, BRG1 knockdown selectively decreased the recruitment of RNA polymerase II to the HO-1 promoter but not to the NQO1 promoter. HO-1, but not other Nrf2-regulated genes, harbors a sequence of TG repeats capable of forming Z-DNA with BRG1 assistance. Similarly, replacement of the TG repeats with an alternative Z-DNA-forming sequence led to BRG1-mediated activation of HO-1. These results thus demonstrate that BRG1, through the facilitation of Z-DNA formation and subsequent recruitment of RNA polymerase II, is critical in Nrf2-mediated inducible expression of HO-1.

Genetic Analysis of Cytoprotective Functions Supported by Graded Expression of Keap1

ABSTRACT Keap1 regulates Nrf2 activity in response to xenobiotic and oxidative stresses. Nrf2 is an essential regulator of cytoprotective genes. Keap1-null mice are lethal by weaning age due to malnutrition caused by severe hyperkeratosis of the upper digestive tract. Analysis of Keap1::Nrf2 double mutant mice revealed that currently recognizable phenotypes of Keap1-null mice are all attributable to constitutive activation of Nrf2. We previously reported that hepatocyte-specific Keap1 knockout (Keap1flox/−::Albumin-Cre) mice are viable and more resistant to acute toxicity of acetaminophen (APAP). In the current study, we found that the floxed Keap1 allele is hypomorphic and that Keap1 expression was decreased in all examined tissues of Keap1flox/− mice. Taking advantage of the hypomorphic phenotype of Keap1flox/− mice, we examined the effects of graded reduction of Keap1 expression in adult mice. When challenged with APAP, Keap1flox/− mice were more protected from mortality than wild-type and even Keap1flox/−::Albumin-Cre mice. In contrast, a decrease in Keap1 levels to less than 50% resulted in increased mortality in a study of 2-year-old mice. These results support our contention that the benefits of Nrf2 activation in acute toxicity are hormetic and that constitutive Nrf2 activation beyond a certain threshold is rather disadvantageous to long-term survival.

Induction of Drug-Metabolizing Enzymes by Garlic and Allyl Sulfide Compounds via Activation of Constitutive Androstane Receptor and Nuclear Factor E2-Related Factor 2

Garlic oil (GO) contains several linear sulfur compounds, including diallyl sulfide (DAS), diallyl disulfide (DADS), and diallyl trisulfide (DATS), that induce drug-metabolizing enzymes such as CYP2B and NAD(P)H quinone oxidoreductase 1 (NQO1). CYP2B and NQO1 are primarily regulated by constitutive androstane receptor (CAR) and nuclear factor E2-related factor 2 (Nrf2) transcription factors, respectively. The purpose of this study was to determine whether GO and its specific constituents induce these two enzymes via CAR and Nrf2 activation. Female Wistar-Kyoto (WKY) rats express little CAR protein and exhibit less induction of CYP2B1/2 than males. GO, DAS, and DADS, but not DATS, induced CYP2B1/2 mRNA levels to a greater extent in WKY males than in females, suggesting CAR activation. Conversely, DAS induced NQO1 levels equally in WKY males and females, indicating CAR-independent induction in rats. DAS, but not GO, DADS, or DATS, induced CYP2B10 mRNA levels 530-fold in wild-type (WT) mice, whereas this induction was attenuated in CAR-/- mice. DAS induced NQO1 in WT and CAR-/- mice equally, suggesting CAR-independent induction in mice. DAS induced NQO1 5-fold in WT mice, whereas induction was completely absent in Nrf2-/- mice, indicating DAS also activates Nrf2. DAS induction of CYP2B10 mRNA was independent of Nrf2 presence or absence. In in vivo transcription assays, DAS activated the human CYP2B6 promoter, and the antioxidant response element of the human NQO1 promoter, respectively. These studies indicate that GO constituents, particularly DAS, activate CAR and Nrf2 to induce drug-metabolizing enzymes.

REGULATION OF MOUSE ORGANIC ANION-TRANSPORTING POLYPEPTIDES (OATPS) IN LIVER BY PROTOTYPICAL MICROSOMAL ENZYME INDUCERS THAT ACTIVATE DISTINCT TRANSCRIPTION FACTOR PATHWAYS

Drug-metabolizing enzymes and transporters are key factors that affect disposition of xenobiotics. Phase I enzyme induction by classes of microsomal enzyme inducers occurs via activation of transcription factors such as aryl hydrocarbon receptor (AhR), constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor α (PPARα), and nuclear factor erythroid 2-related factor 2 (Nrf2). However, regulation of organic anion-transporting polypeptide (Oatp) uptake transporters by these factors is poorly understood. Hepatic Oatp uptake of some chemicals must occur prior to biotransformation; thus, we hypothesize that expression of Oatps and biotransformation enzymes is coordinately regulated in liver. In the present study, the effects of known chemical activators of AhR, CAR, PXR, PPARα, and Nrf2 on the hepatic mRNA expression of mouse Oatps and drug-metabolizing enzymes were quantified by the branched DNA assay. All chemicals increased the expression of their well characterized target drug-metabolizing enzymes: AhR ligands increased Cyp1A1, CAR activators increased Cyp2B10, PXR ligands increased Cyp3A11, PPARα ligands increased Cyp4A14, and Nrf2 activators induced NAD(P)H:quinone oxidoreductase 1. AhR ligands (2,3,7,8-tetrachlorodibenzo-p-dioxin, polychlorinated biphenyl 126, and β-naphthoflavone) increased Oatp2b1 and 3a1 mRNA expression in liver. CAR activators [phenobarbital, 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene, and diallyl sulfide] decreased Oatp1a1 mRNA expression. Two PXR ligands [pregnenolone-16α-carbonitrile (PCN) and spironolactone] increased Oatp1a4 mRNA expression in liver, whereas PXR ligands (PCN, spironolactone, and dexamethasone) and PPARα ligands (clofibrate, ciprofibrate, and diethylhexylphthalate) decreased Oatp1a1, 1b2, 2a1, and 2b1 mRNA expression in liver. Nrf2 activators (oltipraz, ethoxyquin, and butylated hydroxyanisole) down-regulated Oatp1a1 but up-regulated Oatp2b1 mRNA expression. Therefore, only a few transcription factor activators increased Oatp expression, and, surprisingly, many decreased Oatp expression.

Ursodeoxycholic acid stimulates Nrf2-mediated hepatocellular transport, detoxification, and antioxidative stress systems in mice

The protective action of ursodeoxycholic acid (UDCA) in cholestatic liver diseases may be mediated by choleresis, detoxification, and cytoprotection against oxidative stress. Nrf2, one transcription factor, serves as a cellular stress sensor and is a key regulator for hepatic induction of detoxifying enzymes, antioxidative stress genes, and numerous Mrp family members. We aimed to investigate whether UDCA induces hepatic Mrp expression along with that of detoxifying enzymes and antioxidative stress genes via the Nrf2 transcriptional pathway. The protein level, subcellular localization, and mRNA level of Mrp family members were assessed in livers of Keap1 gene-knockdown (Keap1-kd) mice and those of UDCA-fed wild-type (WT) and Nrf2 gene-null (Nrf2-null) mice. Nuclear levels of Nrf2 in livers of Keap1-kd mice markedly increased, resulting in constitutive activation of Nrf2. Keap1-kd mice have high-level expression of hepatic Mrp2, Mrp3, and Mrp4 relative to WT mice. UDCA potently increased nuclear Nrf2 expression level in livers of WT mice, and the treatment showed maximal hepatic induction of Mrp2, Mrp3, and Mrp4 in association with enhanced membranous localizations in an Nrf2-dependent manner. UDCA similarly increased nuclear Nrf2 expression level in rat hepatocytes. Chromatin immunoprecipitation assays using mouse hepatocytes revealed the binding of Nrf2 to antioxidant response elements in the promoter regions of Mrp2, Mrp3, and Mrp4. These findings demonstrate an important role of Nrf2 in the induction of Mrp family members in livers and suggest that a therapeutic mechanism of UDCA action is, via Nrf2 activation, a stimulation of detoxification and antioxidative stress systems, along with Mrp-mediated efflux transport.

Nrf2-deficiency creates a responsive microenvironment for metastasis to the lung

The Nrf2 transcription factor is crucial for regulating the cellular defense against various carcinogens. However, relationship between host Nrf2 and cancer metastasis remains unexplored. To address this issue, we examined susceptibility of Nrf2-deficient mice to pulmonary cancer metastasis following implantation of the mouse Lewis lung carcinoma (3LL) cell line. Nrf2-deficient mice reproducibly exhibited a higher number of pulmonary metastatic nodules than wild-type mice did. The lung and bone marrow (BM) of cancer-bearing Nrf2-deficient mice contained increased numbers of inflammatory cells, including myeloid-derived suppressor cells (MDSCs), a potent population of immunosuppressive cells. MDSCs can attenuate CD8(+) T-cell immunity through modification of the T-cell receptor complex exploiting reactive oxygen species (ROS). MDSCs of Nrf2-deficient mice retained elevated levels of ROS relative to wild-type mice. BM transplantation experiments revealed functional disturbance in the hematopoietic and immune systems of Nrf2-deficient mice. Wild-type recipient mice with Nrf2-deficient BM cells showed increased levels of lung metastasis after cancer cell inoculation. These mice exhibited high-level accumulation of ROS in MDSCs, which showed very good coincidence to the decrease of splenic CD8(+) T-cells. In contrast, Keap1-knockdown mutant mice harboring high-level Nrf2 expression displayed increased resistance against the cancer cell metastasis to the lung, accompanied by a decrease in ROS in the MDSCs fraction. Our results thus reveal a novel function for Nrf2 in the prevention of cancer metastasis, presumably by its ability to preserve the redox balance in the hematopoietic and immune systems.

Nrf2- and PPARα-Mediated Regulation of Hepatic Mrp Transporters after Exposure to Perfluorooctanoic Acid and Perfluorodecanoic Acid

Perfluorooctanoic acid and perfluorodecanoic acid (PFDA) are commonly used as emulsifiers and surfactants in fluoropolymer manufacturing and are known peroxisome proliferator-activated receptor alpha (PPAR alpha) agonists. PPAR alpha activation induces beta- and omega-oxidation enzymes such as Cyp4a14 and acyl-CoA oxidase, which are a likely cause of subsequent oxidative stress and peroxisome proliferation. Conversely, NF-E2-related factor-2 (Nrf2) is a transcription factor that protects against oxidative stress and inflammation by regulating several detoxification and xenobiotic transporter genes. Because PFDA markedly induces hepatic metabolism and oxidative stress, we hypothesized that PFDA exposure would increase expression of hepatic efflux multidrug resistance-associated protein (Mrp) transporters. A single PFDA dose (80 mg/kg) administered to mice increased hepatic Mrp3 (fourfold) and Mrp4 (31-fold) mRNA expression. Upregulation of Mrp3 and Mrp4 correlated with elevated serum-conjugated bilirubin and bile acids, respectively. To determine the mechanism of Mrp3 and Mrp4 induction, PFDA was administered to Nrf2-null mice, PPAR alpha-null mice, and mice pretreated with gadolinium chloride, a Kupffer cell-depleting chemical capable of inhibiting the inflammatory cytokine response. In both PPAR alpha- and Nrf2-null mice, maximal induction of Mrp3 and Mrp4 mRNA after PFDA administration was attenuated. Gadolinium chloride pretreatment reduced serum and hepatic tumor necrosis factor-alpha levels after PFDA treatment, as well as Mrp4 mRNA expression by 30%, suggesting that Kupffer cell-derived mediators may contribute to Mrp induction. Thus, after PFDA administration, the liver mounts a compensatory hepatoprotective response via PPAR alpha and Nrf2, markedly increasing Mrp3 and Mrp4 expression, with corresponding increases in serum of known Mrp3 and Mrp4 substrates.

Nrf2 regulates ferroportin 1-mediated iron efflux and counteracts lipopolysaccharide-induced ferroportin 1 mRNA suppression in macrophages

Iron is an essential element of hemoglobin, and efficient iron recycling from senescent erythrocytes by splenic macrophages is required for erythrocyte hemoglobin synthesis during erythropoiesis. Ferroportin 1 (Fpn1) is the sole iron exporter in mammals, and it also regulates iron reutilization. In this study, we demonstrated genetically that a redox-sensitive transcription factor, Nrf2, regulates Fpn1 mRNA expression in macrophages. Nrf2 activation by several electrophilic compounds commonly resulted in the upregulation of Fpn1 mRNA in bone marrow-derived and peritoneal macrophages obtained from wild-type mice but not from Nrf2 knockout mice. Further, Nrf2 activation enhanced iron release from the J774.1 murine macrophage cell line. Previous studies showed that inflammatory stimuli, such as LPS, downregulates macrophage Fpn1 by transcriptional and hepcidin-mediated post-translational mechanisms leading to iron sequestration by macrophages. We showed that two Nrf2 activators, diethyl maleate and sulforaphane (SFN; a natural Nrf2 activator found in broccoli), restored the LPS-induced suppression of Fpn1 mRNA in human and mouse macrophages, respectively. Furthermore, SFN counteracted the LPS-induced increase of Hepcidin mRNA by an Nrf2-independent mechanism in mouse peritoneal macrophages. These results demonstrate that Nrf2 regulates iron efflux from macrophages through Fpn1 gene transcription and suggest that Nrf2 may control iron metabolism during inflammation.