Ana I. Rojo

Regulation of Heme Oxygenase-1 Expression through the Phosphatidylinositol 3-Kinase/Akt Pathway and the Nrf2 Transcription Factor in Response to the Antioxidant Phytochemical Carnosol

The phosphatidylinositol 3-kinase (PI3K)/Akt pathway elicits a survival signal against multiple apoptotic insults. In addition, phase II enzymes such as heme oxygenase-1 (HO-1) protect cells against diverse toxins and oxidative stress. In this work, we describe a link between these defense systems at the level of transcriptional regulation of the antioxidant enzyme HO-1. The herb-derived phenol carnosol induced HO-1 expression at both mRNA and protein levels. Luciferase reporter assays indicated that carnosol targeted the mouse ho1 promoter at two enhancer regions comprising the antioxidant response elements (AREs). Moreover, carnosol increased the nuclear levels of Nrf2, a transcription factor governing AREs. Electrophoretic mobility shift assays and luciferase reporter assays with a dominant-negative Nrf2 mutant indicated that carnosol increased the binding of Nrf2 to ARE and induced Nrf2-dependent activation of the ho1 promoter. While investigating the signaling pathways responsible for HO-1 induction, we observed that carnosol activated the ERK, p38, and JNK pathways as well as the survival pathway driven by PI3K. Inhibition of PI3K reduced the increase in Nrf2 protein levels and activation of the ho1 promoter. Expression of active PI3K-CAAX (where A is aliphatic amino acid) was sufficient to activate AREs. The use of dominant-negative mutants of protein kinase Cζ and Akt1, two kinases downstream from PI3K, demonstrated a requirement for active Akt1, but not protein kinase Cζ. Moreover, the long-term antioxidant effect of carnosol was partially blocked by PI3K or HO-1 inhibitors, further demonstrating that carnosol attenuates oxidative stress through a pathway that involves PI3K and HO-1.

The transcription factor Nrf2 is a therapeutic target against brain inflammation

Because chronic neuroinflammation is a hallmark of neurodegenerative diseases and compromises neuron viability, it is imperative to discover pharmacologic targets to modulate the activation of immune brain cells, the microglia. In this study, we identify the transcription factor Nrf2, guardian of redox homeostasis, as such target in a model of LPS-induced inflammation in mouse hippocampus. Nrf2 knockout mice were hypersensitive to the neuroinflammation induced by LPS, as determined by an increase in F4/80 mRNA and protein, indicative of an increase in microglial cells, and in the inflammation markers inducible NO synthase, IL-6, and TNF-α, compared with the hippocampi of wild-type littermates. The aliphatic isothiocyanate sulforaphane elicited an Nrf2-mediated antioxidant response in the BV2 microglial cell line, determined by flow cytometry of cells incubated with the redox sensitive probe dihydrodichlorofluorescein diacetate, and by the Nrf2-dependent induction of the phase II antioxidant enzyme heme oxygenase-1. Animals treated with sulforaphane displayed a 2–3-fold increase in heme oxygenase-1, a reduced abundance of microglial cells in the hippocampus and an attenuated production of inflammation markers (inducible NO synthase, IL-6, and TNF-α) in response to LPS. Considering that release of reactive oxygen species is a property of activated microglia, we propose a model in which late induction of Nrf2 intervenes in the down-regulation of microglia. This study opens the possibility of targeting Nrf2 in brain as a means to modulate neuroinflammation.

SCF/β-TrCP promotes glycogen synthase kinase 3-dependent degradation of the Nrf2 transcription factor in a keap1-independent manner

ABSTRACT Regulation of transcription factor Nrf2 (NF-E2-related factor 2) involves redox-sensitive proteasomal degradation via the E3 ubiquitin ligase Keap1/Cul3. However, Nrf2 is controlled by other mechanisms that have not yet been elucidated. We now show that glycogen synthase kinase 3 (GSK-3) phosphorylates a group of Ser residues in the Neh6 domain of mouse Nrf2 that overlap with an SCF/β-TrCP destruction motif (DSGIS, residues 334 to 338) and promotes its degradation in a Keap1-independent manner. Nrf2 was stabilized by GSK-3 inhibitors in Keap1-null mouse embryo fibroblasts. Similarly, an Nrf2ΔETGE mutant, which cannot be degraded via Keap1, accumulated when GSK-3 activity was blocked. Phosphorylation of a Ser cluster in the Neh6 domain of Nrf2 stimulated its degradation because a mutant Nrf2ΔETGE 6S/6A protein, lacking these Ser residues, exhibited a longer half-life than Nrf2ΔETGE. Moreover, Nrf2ΔETGE 6S/6A was insensitive to β-TrCP regulation and exhibited lower levels of ubiquitination than Nrf2ΔETGE. GSK-3β enhanced ubiquitination of Nrf2ΔETGE but not that of Nrf2ΔETGE 6S/6A. The Nrf2ΔETGE protein but not Nrf2ΔETGE 6S/6A coimmunoprecipitated with β-TrCP, and this association was enhanced by GSK-3β. Our results show for the first time that Nrf2 is targeted by GSK-3 for SCF/β-TrCP-dependent degradation. We propose a “dual degradation” model to describe the regulation of Nrf2 under different pathophysiological conditions.

Glycogen Synthase Kinase-3β Inhibits the Xenobiotic and Antioxidant Cell Response by Direct Phosphorylation and Nuclear Exclusion of the Transcription Factor Nrf2

The transcription factor Nrf2 (nuclear factor E2-related factor 2) regulates the expression of antioxidant phase II genes and contributes to preserve redox homeostasis and cell viability in response to oxidant insults. Nrf2 should be coordinated with the canonical cell survival pathway represented by phosphatidylinositol 3-kinase (PI3K) and the Ser/Thr kinase Akt but so far the mechanistic connections remain undefined. Here we identify glycogen synthase kinase-3β (GSK-3β), which is inhibited by Akt-mediated phosphorylation, as the link between both processes. Using heme oxygenase-1 (HO-1) as a model phase II gene, we found that both PI3K and Akt increased mRNA and protein levels of this enzyme. Pharmacological inhibitors (LiCl and PDZD-8) and genetic variants of GSK-3β (constitutively active and dominant negative mutants) indicated that PI3K/Akt activates and GSK-3β inhibits the antioxidant response elements of the ho1 promoter and pointed Nrf2 as directly involved in this process. Indeed, GSK-3β phosphorylated Nrf2 in vitro and in vivo. Immunocytochemistry and subcellular fractionation analyses demonstrated that the effect of GSK-3β-mediated phosphorylation of Nrf2 is to exclude this transcription factor from the nucleus. Nrf2 up-regulated the expression of HO-1, glutathione peroxidase, glutathione S-transferase A1, NAD(P)H: quinone oxidoreductase and glutamate-cysteine ligase and protected against hydrogen peroxide-induced glutathione depletion and cell death, whereas co-expression of active GSK-3β attenuated both phase II gene expression and oxidant protection. These results contribute to clarify the cross-talk between the survival signal elicited by PI3K/Akt and the antioxidant phase II cell response, and introduce GSK-3β as the key mediator of this regulation mechanism.

Nrf2 regulates microglial dynamics and neuroinflammation in experimental Parkinson's disease.

Neural injury leads to inflammation and activation of microglia that in turn may participate in progression of neurodegeneration. The mechanisms involved in changing microglial activity from beneficial to chronic detrimental neuroinflammation are not known but reactive oxygen species (ROS) may be involved. We have addressed this question in Nrf2‐knockout mice, with hypersensitivity to oxidative stress, submitted to daily inoculation of 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) for 4 weeks. Basal ganglia of these mice exhibited a more severe dopaminergic dysfunction than wild type littermates in response to MPTP. The amount of CD11b‐positive/CD45‐highly‐stained cells, indicative of peripheral macrophage infiltration, did not increase significantly in response to MPTP. However, Nrf2‐deficient mice exhibited more astrogliosis and microgliosis as determined by an increase in messenger RNA and protein levels for GFAP and F4/80, respectively. Inflammation markers characteristic of classical microglial activation, COX‐2, iNOS, IL‐6, and TNF‐α were also increased and, at the same time, anti‐inflammatory markers attributable to alternative microglial activation, such as FIZZ‐1, YM‐1, Arginase‐1, and IL‐4 were decreased. These results were confirmed in microglial cultures stimulated with apoptotic conditioned medium from MPP+‐treated dopaminergic cells, further demonstrating a role of Nrf2 in tuning balance between classical and alternative microglial activation. This study demonstrates a crucial role of Nrf2 in modulation of microglial dynamics and identifies Nrf2 as molecular target to control microglial function in Parkinsons disease (PD) progression.

Regulation of Cu/Zn-Superoxide Dismutase Expression via the Phosphatidylinositol 3 Kinase/Akt Pathway and Nuclear Factor-κB

Aerobic cells adjust the expression of antioxidant enzymes to maintain reactive oxygen species within tolerable levels. In addition, phosphatidylinositol 3 kinase (PI3K) and its downstream protein kinase effector Akt adapt cells to survive in the presence of oxidative stress. Here we provide evidence for an association between these two defense systems via transcriptional regulation of Cu/Zn-superoxide dismutase (Cu/Zn-SOD). PC12 pheochromocytoma cells expressing active Akt1 exhibit lower ROS levels in response to hydrogen peroxide, as determined with the superoxide-sensitive probe hydroethidine. Transfection of constitutive or 4-hydroxytamoxifen-inducible versions of Akt1 results in higher messenger RNA and protein levels of Cu/Zn-SOD. Luciferase reporter constructs, carrying different length fragments of the human sod1 gene promoter, have identified a region between -552 and -355 that is targeted by PI3K and Akt and that contains a putative site of regulation by nuclear factor-κB (NF-κB). Nerve growth factor (NGF) and Akt augment the transactivating activity and produce higher nuclear levels of p65-NF-κB. Electrophoretic mobility shift assays indicate that the putative NF-κB regulatory sequence binds p65-NF-κB more efficiently in nuclear extracts from these cells. A dominant-negative mutant of IκBα further demonstrates that the PI3K/Akt axis targets the sod1 promoter at the level of the newly characterized NF-κB site. These results illustrate a new mechanism by which the PI3K/Akt pathway protects cells against oxidative stress, involving the upregulation of Cu/Zn-SOD gene expression, and the results identify NF-κB as a key mediator in the regulation of this gene.

GSK-3β down-regulates the transcription factor Nrf2 after oxidant damage: relevance to exposure of neuronal cells to oxidative stress

Oxidant injury activates the neuroprotective pathway represented by phosphatidylinositol 3 kinase (PI3K) and Akt. However, the final outcome of oxidant exposure is often associated with neuronal death. This study was aimed to identify the molecular mechanism responsible for loss of tolerance to an oxidative environment. In N2A neuroblasts, serum and H2O2 exhibited different kinetics of regulation for the Ser/Thr kinases Akt and glycogen synthase kinase 3β (GSK‐3β) and for the transcription factor Nrf2, which governs redox homeostasis. Thus, H2O2 rapidly activated Akt, inhibited GSK‐3β, and directed the transcription factor Nrf2 to the nucleus, but after 4 h Akt was inactive, GSK‐3β was active and Nrf2 was more cytosolic than nuclear. Inhibition of the PI3K/Akt pathway by LY294002, impeded the short‐term effect of H2O2 on nuclear translocation of Nrf2. GSK‐3β activation (inhibiting PI3K/Akt) or direct GSK‐3β inhibition in cerebellar granule neurons resulted in respective nuclear exclusion and nuclear accumulation of Nrf2. Moreover, in these neurons, nuclear accumulation of Nrf2 correlated with increased heme oxygenase‐1 expression. Over‐expression of the kinase active GSK‐3β (Δ9) mutant, induced Nrf2 cytoplasmic localization and inhibited Nrf2 transcriptional activity towards an antioxidant‐response‐element luciferase reporter. Moreover, GSK‐3β (Δ9) sensitized N2A neuroblasts to H2O2‐induced oxidative stress and cell death. This study identifies GSK‐3β, a kinase known to participate in neurodegeneration, as a fundamental element in the down‐regulation of the antioxidant cell defense elicited by Nrf2 after oxidant injury and provides a mechanism to explain the loss of oxidant tolerance that happens under persistent oxidant exposure such as those found in several neuropathologies.

Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

ABSTRACT The transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and β-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3β exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Pharmacological targeting of the transcription factor Nrf2 at the basal ganglia provides disease modifying therapy for experimental parkinsonism

Current therapies for motor symptoms of Parkinsons disease (PD) are based on dopamine replacement. However, the disease progression remains unaffected, because of continuous dopaminergic neuron loss. Since oxidative stress is actively involved in neuronal death in PD, pharmacological targeting of the antioxidant machinery may have therapeutic value. Here, we analyzed the relevance of the antioxidant phase II response mediated by the transcription factor NF-E2-related factor 2 (Nrf2) on brain protection against the parkinsonian toxin methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Intraperitoneal administration of the potent Nrf2 activator sulforaphane (SFN) increased Nrf2 protein levels in the basal ganglia and led to upregulation of phase II antioxidant enzymes heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase (NQO1). In wild-type mice, but not in Nrf2-knockout mice, SFN protected against MPTP-induced death of nigral dopaminergic neurons. The neuroprotective effects were accompanied by a decrease in astrogliosis, microgliosis, and release of pro-inflammatory cytokines. These results provide strong pharmacokinetic and biochemical evidence for activation of Nrf2 and phase II genes in the brain and also offer a neuroprotective strategy that may have clinical relevance for PD therapy.

Heme Oxygenase-1 as a Therapeutic Target in Neurodegenerative Diseases and Brain Infections

Heme oxygenase-1 (HO-1) catalyzes the degradation of heme to generate carbon monoxide, biliverdin and free iron. Increased HO-1 levels constitute an anatomopathological feature of many neurological diseases, such as neurodegenerative disorders and brain infections, which correlate with exacerbated oxidative stress and inflammation. It is generally accepted that the elevated HO-1 levels represent an attempt to restore redox homeostasis and to down-modulate inflammation. However, experimental observations indicate that the extent of HO-1 induction may be critical because excessive heme degradation may result in toxic levels of CO, bilirubin and, more importantly, iron. Pharmacological modulation of HO-1 levels in the brain, within therapeutic limits, shows promising results in models of Alzheimers (AD), Parkinsons (PD) and of infectious diseases, such as malaria. A more complete understanding on how HO-1 is involved in the pathogenesis of neurological diseases will be essential to develop therapeutic approaches. In the next coming years we will witness the description of chemicals, drugs or dietary products that cross the blood brain barrier efficiently, activate HO-1 expression, and achieve neuroprotective and anti-inflammatory effects in vivo.