An inhibitor of interaction between the transcription factor NRF2 and the E3 ubiquitin ligase adapter ß-TrCP delivers anti-inflammatory responses in mouse liver.

It is widely accepted that activating the transcription factor NRF2 will blast the physiological anti-inflammatory mechanisms, which will help combat pathologic inflammation. Much effort is being put in inhibiting the main NRF2 repressor, KEAP1, with either electrophilic small molecules or disrupters of the KEAP1/NRF2 interaction. However, targeting ß-TrCP, the non-canonical repressor of NRF2, has not been considered yet. After in silico screening of ∼1 million compounds, we now describe a novel small molecule, PHAR, that selectively inhibits the interaction between ß-TrCP and the phosphodegron in transcription factor NRF2. PHAR upregulates NRF2-target genes such as Hmox1, Nqo1, Gclc, Gclm and Aox1, in a KEAP1-independent, but ß-TrCP dependent manner, breaks the ß-TrCP/NRF2 interaction in the cell nucleus, and inhibits the ß-TrCP-mediated in vitro ubiquitination of NRF2. PHAR attenuates hydrogen peroxide induced oxidative stress and, in lipopolysaccharide-treated macrophages, it downregulates the expression of inflammatory genes Il1b, Il6, Cox2, Nos2. In mice, PHAR selectively targets the liver and greatly attenuates LPS-induced liver inflammation as indicated by a reduction in the gene expression of the inflammatory cytokines Il1b, TNf, and Il6, and in F4/80-stained liver resident macrophages. Thus, PHAR offers a still unexplored alternative to current NRF2 activators by acting as a ß-TrCP/NRF2 interaction inhibitor that may have a therapeutic value against undesirable inflammation.

Nordihydroguaiaretic Acid: From Herbal Medicine to Clinical Development for Cancer and Chronic Diseases.

Nordihydroguaiaretic acid (NDGA) is a phenolic lignan obtained from Larrea tridentata, the creosote bush found in Mexico and USA deserts, that has been used in traditional medicine for the treatment of numerous diseases such as cancer, renal, cardiovascular, immunological, and neurological disorders, and even aging. NDGA presents two catechol rings that confer a very potent antioxidant activity by scavenging oxygen free radicals and this may explain part of its therapeutic action. Additional effects include inhibition of lipoxygenases (LOXs) and activation of signaling pathways that impinge on the transcription factor Nuclear Factor Erythroid 2-related Factor (NRF2). On the other hand, the oxidation of the catechols to the corresponding quinones my elicit alterations in proteins and DNA that raise safety concerns. This review describes the current knowledge on NDGA, its targets and side effects, and its synthetic analogs as promising therapeutic agents, highlighting their mechanism of action and clinical projection towards therapy of neurodegenerative, liver, and kidney disease, as well as cancer.

Transcription factor NRF2 uses the Hippo pathway effector TAZ to induce tumorigenesis in glioblastomas.

Transcription factor NRF2 orchestrates a cellular defense against oxidative stress and, so far, has been involved in tumor progression by providing a metabolic adaptation to tumorigenic demands and resistance to chemotherapeutics. In this study, we discover that NRF2 also propels tumorigenesis in gliomas and glioblastomas by inducing the expression of the transcriptional co-activator TAZ, a protein of the Hippo signaling pathway that promotes tumor growth. The expression of the genes encoding NRF2 (NFE2L2) and TAZ (WWTR1) showed a positive correlation in 721 gliomas from The Cancer Genome Atlas database. Moreover, NRF2 and TAZ protein levels also correlated in immunohistochemical tissue arrays of glioblastomas. Genetic knock-down of NRF2 decreased, while NRF2 overexpression or chemical activation with sulforaphane, increased TAZ transcript and protein levels. Mechanistically, we identified several NRF2-regulated functional enhancers in the regulatory region of WWTR1. The relevance of the new NRF2/TAZ axis in tumorigenesis was demonstrated in subcutaneous and intracranial grafts. Thus, intracranial inoculation of NRF2-depleted glioma stem cells did not develop tumors as determined by magnetic resonance imaging. Forced TAZ overexpression partly rescued both stem cell growth in neurospheres and tumorigenicity. Hence, NRF2 not only enables tumor cells to be competent to proliferate but it also propels tumorigenesis by activating the TAZ-mediated Hippo transcriptional program.

Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases.

The transcription factor NF-E2 p45-related factor 2 (NRF2; encoded by NFE2L2) and its principal negative regulator, the E3 ligase adaptor Kelch-like ECH-associated protein 1 (KEAP1), are critical in the maintenance of redox, metabolic and protein homeostasis, as well as the regulation of inflammation. Thus, NRF2 activation provides cytoprotection against numerous pathologies including chronic diseases of the lung and liver; autoimmune, neurodegenerative and metabolic disorders; and cancer initiation. One NRF2 activator has received clinical approval and several electrophilic modifiers of the cysteine-based sensor KEAP1 and inhibitors of its interaction with NRF2 are now in clinical development. However, challenges regarding target specificity, pharmacodynamic properties, efficacy and safety remain.

Transcription Factor NRF2 as a Therapeutic Target for Chronic Diseases: A Systems Medicine Approach.

Systems medicine has a mechanism-based rather than a symptom- or organ-based approach to disease and identifies therapeutic targets in a nonhypothesis-driven manner. In this work, we apply this to transcription factor nuclear factor (erythroid-derived 2)-like 2 (NRF2) by cross-validating its position in a protein-protein interaction network (the NRF2 interactome) functionally linked to cytoprotection in low-grade stress, chronic inflammation, metabolic alterations, and reactive oxygen species formation. Multiscale network analysis of these molecular profiles suggests alterations of NRF2 expression and activity as a common mechanism in a subnetwork of diseases (the NRF2 diseasome). This network joins apparently heterogeneous phenotypes such as autoimmune, respiratory, digestive, cardiovascular, metabolic, and neurodegenerative diseases, along with cancer. Importantly, this approach matches and confirms in silico several applications for NRF2-modulating drugs validated in vivo at different phases of clinical development. Pharmacologically, their profile is as diverse as electrophilic dimethyl fumarate, synthetic triterpenoids like bardoxolone methyl and sulforaphane, protein-protein or DNA-protein interaction inhibitors, and even registered drugs such as metformin and statins, which activate NRF2 and may be repurposed for indications within the NRF2 cluster of disease phenotypes. Thus, NRF2 represents one of the first targets fully embraced by classic and systems medicine approaches to facilitate both drug development and drug repurposing by focusing on a set of disease phenotypes that appear to be mechanistically linked. The resulting NRF2 drugome may therefore rapidly advance several surprising clinical options for this subset of chronic diseases.

Discovery of the first dual GSK3ß inhibitor/Nrf2 inducer. A new multitarget therapeutic strategy for Alzheimer's disease.

The formation of neurofibrillary tangles (NFTs), oxidative stress and neuroinflammation have emerged as key targets for the treatment of Alzheimer's disease (AD), the most prevalent neurodegenerative disorder. These pathological hallmarks are closely related to the over-activity of the enzyme GSK3ß and the downregulation of the defense pathway Nrf2-EpRE observed in AD patients. Herein, we report the synthesis and pharmacological evaluation of a new family of multitarget 2,4-dihydropyrano[2,3-c]pyrazoles as dual GSK3ß inhibitors and Nrf2 inducers. These compounds are able to inhibit GSK3ß and induce the Nrf2 phase II antioxidant and anti-inflammatory pathway at micromolar concentrations, showing interesting structure-activity relationships. The association of both activities has resulted in a remarkable anti-inflammatory ability with an interesting neuroprotective profile on in vitro models of neuronal death induced by oxidative stress and energy depletion and AD. Furthermore, none of the compounds exhibited in vitro neurotoxicity or hepatotoxicity and hence they had improved safety profiles compared to the known electrophilic Nrf2 inducers. In conclusion, the combination of both activities in this family of multitarget compounds confers them a notable interest for the development of lead compounds for the treatment of AD.

The PTEN/NRF2 axis promotes human carcinogenesis.

AIMS: A recent study conducted in mice reported that liver-specific knockout of tumor suppressor Pten augments nuclear factor (erythroid-derived 2)-like 2 (NRF2) transcriptional activity. Here, we further investigated how phosphatase and tensin homolog deleted on chromosome 10 (PTEN) controls NRF2 and the relevance of this pathway in human carcin ogenesis. RESULTS: Drug and genetic targeting to PTEN and phosphoproteomics approaches indicated that PTEN leads to glycogen synthase kinase-3 (GSK-3)-mediated phosphorylation of NRF2 at residues Ser(335) and Ser(338) and subsequent beta-transducin repeat containing protein (ß-TrCP)-dependent but Kelch-like ECH-associated protein 1 (KEAP1)-independent degradation. Rescue experiments in PTEN-deficient cells and xerographs in athymic mice indicated that loss of PTEN leads to increased NRF2 signature which provides a proliferating and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). INNOVATION: These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3/ß-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. CONCLUSION: Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target.

Redox control of microglial function: molecular mechanisms and functional significance.

Neurodegenerative diseases are characterized by chronic microglial over-activation and oxidative stress. It is now beginning to be recognized that reactive oxygen species (ROS) produced by either microglia or the surrounding environment not only impact neurons but also modulate microglial activity. In this review, we first analyze the hallmarks of pro-inflammatory and anti-inflammatory phenotypes of microglia and their regulation by ROS. Then, we consider the production of reactive oxygen and nitrogen species by NADPH oxidases and nitric oxide synthases and the new findings that also indicate an essential role of glutathione (γ-glutamyl-l-cysteinylglycine) in redox homeostasis of microglia. The effect of oxidant modification of macromolecules on signaling is analyzed at the level of oxidized lipid by-products and sulfhydryl modification of microglial proteins. Redox signaling has a profound impact on two transcription factors that modulate microglial fate, nuclear factor kappa-light-chain-enhancer of activated B cells, and nuclear factor (erythroid-derived 2)-like 2, master regulators of the pro-inflammatory and antioxidant responses of microglia, respectively. The relevance of these proteins in the modulation of microglial activity and the interplay between them will be evaluated. Finally, the relevance of ROS in altering blood brain barrier permeability is discussed. Recent examples of the importance of these findings in the onset or progression of neurodegenerative diseases are also discussed. This review should provide a profound insight into the role of redox homeostasis in microglial activity and help in the identification of new promising targets to control neuroinflammation through redox control of the brain.

Nuclear import and export signals control the subcellular localization of Nurr1 protein in response to oxidative stress.

Orphan receptor Nurr1 participates in the acquisition and maintenance of the dopaminergic cell phenotype, modulation of inflammation, and cytoprotection, but little is known about its regulation. In this study, we report that Nurr1 contains a bipartite nuclear localization signal (NLS) within its DNA binding domain and two leucine-rich nuclear export signals (NES) in its ligand binding domain. Together, these signals regulate Nurr1 shuttling in and out of the nucleus. Immunofluorescence and immunoblot analysis revealed that Nurr1 is mostly nuclear. A Nurr1 mutant lacking the NLS failed to enter the nucleus. The Nurr1 NLS sequence, when fused to green fluorescent protein, led to nuclear accumulation of this chimeric protein, indicating that this sequence was sufficient to direct nuclear localization of Nurr1. Furthermore, two NES were characterized in the ligand binding domain, whose deletion caused Nurr1 to accumulate predominantly in the nucleus. The Nurr1 NES was sensitive to CRM1 and could function as an independent export signal when fused to green fluorescent protein. Sodium arsenite, an agent that induces oxidative stress, promoted nuclear export of ectopically expressed Nurr1 in HEK293T cells, and the antioxidant N-acetylcysteine rescued from this effect. Similarly, in dopaminergic MN9D cells, arsenite induced the export of endogenous Nurr1, resulting in the loss of expression of Nurr1-dependent genes. This study illustrates that Nurr1 shuttling between the cytosol and nucleus is controlled by specific nuclear import and export signals and that oxidative stress can unbalance the distribution of Nurr1 to favor its cytosolic accumulation.

Signaling pathways activated by the phytochemical nordihydroguaiaretic acid contribute to a Keap1-independent regulation of Nrf2 stability: Role of glycogen synthase kinase-3.

Defense against oxidative stress is executed by an antioxidant program that is tightly controlled by the transcription factor Nrf2. The stability of Nrf2 involves the interaction of two degradation domains, designated Neh2 and Neh6, with the E3 ubiquitin ligase adaptors, Keap1 and ß-TrCP, respectively. The regulation of Nrf2 through the Neh6 degron remains largely unexplored but requires GSK-3 to form a phosphodegron. In this study, the cancer-chemopreventive agent nordihydroguaiaretic acid (NDGA) increased the level of Nrf2 protein and expression of heme oxygenase-1 (HO-1) in kidney-derived LLC-PK1 and HEK293T cells and in wild-type mouse embryo fibroblasts (MEFs). However, NDGA did not induce HO-1 in Nrf2(-/-) MEFs, indicating that Nrf2 is required for induction. The relevance of the Nrf2/HO-1 axis to antioxidant protection was further demonstrated by the finding that the HO-1 inhibitor stannous-mesoporphyrin abolished protection against hydrogen peroxide conferred by NDGA. NDGA increased Nrf2 and HO-1 protein levels in Keap1(-/-) MEFs, implying that Keap1-independent mechanisms regulate Nrf2 stability. Mutants of the Neh2 or Nrh6 domain and chimeric proteins comprising cyan fluorescent protein fused to Neh2 and green fluorescent protein fused to Neh6 exhibited longer half-lives in the presence of NDGA, demonstrating that NDGA targets both the Neh2 and the Neh6 degrons. In common with other chemopreventive agents, NDGA activated the ERK1/2, p38, JNK, and PI3K pathways. By using selective kinase inhibitors we found that PI3K, JNK, and p38 were responsible for the stabilization of Nrf2 and induction of HO-1 by NDGA. To explain how NDGA might up-regulate Nrf2 in a Keap1-independent manner we explored the participation of GSK-3ß because it controls the Neh6 phosphodegron. Importantly, NDGA caused inhibitory phosphorylation of GSK-3ß at Ser9 and at Thr390, and this was associated with a substantial reduction in Neh6 phosphorylation. Our study demonstrates that NDGA activates Nrf2 through multiple signaling cascades and identifies GSK-3ß as an integrator of these signaling pathways and a gatekeeper of Nrf2 stability at the level of the Neh6 phosphodegron.

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

Current therapies for motor symptoms of Parkinson's 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.

The purinergic P2Y(13) receptor activates the Nrf2/HO-1 axis and protects against oxidative stress-induced neuronal death.

Although extracellular purines may have both trophic and apoptotic functions in the brain depending on the targeted purine receptor and cell type, little is known about the role of specific purine receptors on neurons. In this study, we demonstrate that both ADP and its stable analogue 2-methyl-thio-ADP (2MeSADP) induce up-regulation of the cytoprotective protein heme oxygenase-1 (HO-1). Selective inhibition of 2MeSADP-responsive receptors P2Y(1) and P2Y(13) with their respective antagonists MRS2179 and MRS2211 and the use of pertussis toxin demonstrated a role of the purinergic P2Y(13) receptor in this response. Moreover, luciferase assays demonstrated that ectopic expression of the P2Y(13) receptor in neuroblastoma N2A cells resulted in 2MeSADP-dependent induction of antioxidant response elements from the HO-1 promoter. The transcription factor Nrf2 was critical for HO-1 activation and translocated from the cytosol to the nucleus in response to 2MeSADP. In cerebellar granule neurons (CGNs) derived from Nrf2-knockout mice this purine did not activate the Nrf2/HO-1 axis and did not protect against H(2)O(2)-induced cell death. The relevance of HO-1 in 2MeSADP-induced neuroprotection was further demonstrated by the evidence that HO-1 inhibition with tin protoporphyrin (SnPP) prevented protection against H(2)O(2)-induced oxidative stress and cell death. These observations reveal a previously unrecognized role in protection against oxidative stress by extracellular purines acting on the metabotropic P2Y(13) receptor and provide new perspectives for neuroprotective therapies.

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-alpha 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 Parkinson's disease (PD) progression.

The muscarinic M1 receptor activates Nrf2 through a signaling cascade that involves protein kinase C and inhibition of GSK-3beta: connecting neurotransmission with neuroprotection.

In this study, we provide evidence that the muscarinic M1 receptor targets NF-E2-related factor-2 (Nrf2), a transcription factor that regulates the expression of genes containing antioxidant response elements (AREs) in their promoters and that collectively constitute the phase II antioxidant response. In hippocampal primary and cerebellar granule neuron cultures expressing endogenous M1 receptor, carbachol increased the levels of a prototypical phase II antioxidant enzyme, heme oxygenase-1. Moreover, in a heterologous system, based on lentiviral expression of M1 receptor in PC12 pheochromocytoma cells, we found that M1 increased total and nuclear Nrf2 protein levels and heme oxygenase-1 messenger RNA and protein levels. Luciferase reporter constructs for AREs and the use of two inhibitors of protein kinase C (PKC), chelerythrine and 2-aminoethyl diphenylborinate, or transfection with relevant expression vectors allowed us to identify Galphaq, phospholipase C-beta and the classical PKC-gamma isoenzyme, as responsible for the regulation of Nrf2. A PKC-insensitive Nrf2S40A single-point mutant partially channeled M1 signaling to AREs, therefore suggesting the participation of additional intermediates. Inhibition of glycogen synthase kinase-3beta (GSK-3beta) augmented M1-dependent activation of AREs while a PKC-insensitive mutant of GSK-3beta (GSK-3beta-Delta9) blocked this effect and prevented M1-induced accumulation of Nrf2 in the nucleus. Our results demonstrate a previously unidentified role of the Galphaq/phospholipase C-beta/PKC/GSK-3beta axis in regulation of Nrf2 by M1. Such role provides additional conceptual support for the use of cholinemimetics in the treatment of pathologies that, like Alzheimer's disease, require a reinforcement of the cell antioxidant capacity.

Functional interference between glycogen synthase kinase-3 beta and the transcription factor Nrf2 in protection against kainate-induced hippocampal cell death.

Excitotoxicity mediated by glutamate receptors may underlay the pathology of several neurologic diseases. Considering that oxidative stress is central to excitotoxic damage, in this study we sought to analyze if the transcription factor Nrf2, guardian of redox homeostasis, might be targeted to prevent kainate-induced neuron death. Hippocampal slices from Nrf2 knockout mice exhibited increased oxidative stress and cell death compared to those of control mice in response to kainate, as determined with the redox sensitive probes 2,7-dichlorodihydrofluorescein diacetate (H(2)DCFAC) and propidium iodide and lactate dehydrogenase release, respectively, therefore demonstrating a role of Nrf2 in antioxidant protection against excitotoxicity. In the hippocampus of mice intraperitoneally injected with kainate we observed a rapid activation of Akt, inhibition of GSK-3beta and translocation of Nrf2 to the nucleus, but after 4 h Akt was inactive, GSK-3beta was active and Nrf2 was mostly cytosolic, therefore extending our previous studies which indicate that GSK-3beta excludes Nrf2 from the nucleus. Lithium, a GSK-3beta inhibitor, promoted Nrf2 transcriptional activity towards an Antioxidant-Response-Element (ARE) luciferase reporter and cooperated with sulforaphane (SFN) to induce this reporter and to increase the protein levels of heme oxygenase-1 (HO-1), coded by a representative ARE-containing gene. Conversely, ARE activation by SFN was attenuated by over-expression of active GSK-3beta. Finally, combined treatment with SFN and lithium attenuated oxidative stress and cell death in kainate-treated hippocampal slices of wild type mice but not Nrf2 null littermates. Our findings identify the axis GSK-3beta/Nrf2 as a pharmacological target in prevention of excitotoxic neuronal death.

Regulation of heme oxygenase-1 gene expression through the phosphatidylinositol 3-kinase/PKC-zeta pathway and Sp1.

The molecular mechanisms involved in modulation of the antioxidant cell defence by survival signals remain largely unexplored. Here, we report a mechanistic connection between the survival signal elicited by nerve growth factor (NGF) and the antioxidant cell defence represented by heme oxygenase-1 (HO-1) at the level of a newly identified Sp1 site in the human ho1 proximal promoter. By using luciferase reporter constructs we identified a PI3K-responsive region containing a GC-box that resembled the response element for Sp1. Indeed, transfection of Sp1-deficient SL2 cells, electrophoretic mobility shift assays, the use of the GC-box binding drug mithramycin, and mutation of the GC-box provided evidence for a Sp1-like site in the PI3K-sensitive region. Then, we observed with the use of a Sp1-Gal4 chimera that PI3K regulates the transactivating capacity of Sp1. Cotransfection of active PI3K and PKC-zeta expression vectors resulted in substantial increase of Sp1 phosphorylation and in synergistic activation of both Sp1-Gal4 and endogenous Sp1. Moreover, these effects were mimicked by cotransfection of active MEK and ERK expression vectors and were blocked by the MEK inhibitor PD98059. Inhibition of HO-1 with Sn protoporphyrin IX and blockage of Sp-1-mediatied upregulation of HO-1 with mithramycin attenuated antioxidant and cytoprotective functions of NGF against hydrogen peroxide. This study elucidates how NGF contributes to protection of target cells against oxidative stress.

Glycogen synthase kinase-3beta 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-3beta (GSK-3beta), 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-3beta (constitutively active and dominant negative mutants) indicated that PI3K/Akt activates and GSK-3beta inhibits the antioxidant response elements of the ho1 promoter and pointed Nrf2 as directly involved in this process. Indeed, GSK-3beta phosphorylated Nrf2 in vitro and in vivo. Immunocytochemistry and subcellular fractionation analyses demonstrated that the effect of GSK-3beta-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-3beta 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-3beta as the key mediator of this regulation mechanism.

Protein kinase Akt/PKB phosphorylates heme oxygenase-1 in vitro and in vivo.

Heme oxygenase-1 (HO-1) is a stress response protein that protects cells against diverse noxious stimuli. Although regulation of HO-1 occurs mainly at the transcriptional level, its posttranslational modifications remain unexplored. We have identified a putative consensus sequence for phosphorylation by Akt/PKB of HO-1 at Ser188. Recombinant human and rat HO-1, but not mutant HO-1(S188A), are phosphorylated in vitro by Akt/PKB. Isotopic 32P-labeling of HEK293T cells confirmed that HO-1 is a phosphoprotein and that the basal HO-1 phosphorylation is increased by Akt1 activation. HO-1(S188D), a single point mutant equivalent to the phosphorylated protein, exhibited over 1.6-fold higher activity than wild type HO-1. Fluorescence resonance energy transfer (FRET) studies indicated that HO-1(S188D) bound to cytochrome P450 reductase (CPR) and biliverdin reductase (BVR) with a slightly lower Kd than wild-type HO-1. Although the changes in activity are small, this study provides the first evidence for a role of the survival kinase Akt in the regulation of HO-1.

Regulation of Cu/Zn-superoxide dismutase expression via the phosphatidylinositol 3 kinase/Akt pathway and nuclear factor-kappaB.

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-kappaB (NF-kappaB). Nerve growth factor (NGF) and Akt augment the transactivating activity and produce higher nuclear levels of p65-NF-kappaB. Electrophoretic mobility shift assays indicate that the putative NF-kappaB regulatory sequence binds p65-NF-kappaB more efficiently in nuclear extracts from these cells. A dominant-negative mutant of IkappaBalpha further demonstrates that the PI3K/Akt axis targets the sod1 promoter at the level of the newly characterized NF-kappaB 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-kappaB as a key mediator in the regulation of this gene.

Signaling through the leukocyte integrin LFA-1 in T cells induces a transient activation of Rac-1 that is regulated by Vav and PI3K/Akt-1.

Integrin LFA-1 is a receptor that is able to transmit multiple intracellular signals in leukocytes. Herein we show that LFA-1 induces a potent and transient increase in the activity of the small GTPase Rac-1 in T cells. Maximal Rac-1 activity peaked 10-15 min after LFA-1 stimulation and rapidly declined to basal levels at longer times. We have identified Vav, a guanine nucleotide exchange factor for Rac-1, and PI3K/Akt, as regulators of the activation and inactivation phases of the activity of Rac-1, respectively, in the context of LFA-1 signaling based on the following experimental evidence: (i) LFA-1 induced activation of Vav and PI3K/Akt with kinetics consistent with a regulatory role for these molecules on Rac-1, (ii) overexpression of a constitutively active Vav mutant induces activation of Rac independently of LFA-1 stimulation whereas overexpression of a dominant-negative Vav mutant blocks LFA-1-mediated Rac activation, (iii) pharmacological inhibition of PI3K/Akt prevented the fall in the activity of Rac-1 after its initial activation but had no effect on Vav activity, and (iv) overexpression of a dominant-negative or a constitutively active Akt-1 induced or inhibited, respectively, Rac-1 activity. Finally, we show that T cells with a sustained Rac activity have impaired capacity to elongate onto ICAM-1. These results demonstrate that down-regulation of the activity of this GTPase is a requirement for the regulation of T cell morphology and motility and highlight the importance of temporal regulation of the signaling triggered from this integrin.